Vaccines for HSV-2

ABSTRACT

Compositions of recombinant HSV-2 proteins and an agonist of the innate immune system, such as an adjuvant, are provided as a vaccine. Proteins include an envelope glycoprotein and a structural protein other than an envelope glycoprotein, e.g., a capsid or tegument protein. The vaccine is for use in either HSV-2 seropositive or seronegative subjects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application Nos. 61/647,764, filed May 16, 2012,61/679,387, filed Aug. 3, 2012, and 61/714,158, filed Oct. 15, 2012, allof which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

The sequence listing of this patent application is provided separatelyin a file named “47733_SeqListing.txt”. The content of this file, whichwas created on 16 May 2013, and consists of 45,969 bytes, isincorporated in its entirety.

TECHNICAL FIELD

Vaccines for herpes simplex virus-2 infection and related methods andcompositions.

BACKGROUND

HSV-2 (herpes simplex virus-2) is a member of the family Herpetoviridae,a group of DNA viruses that often result in skin lesions (e.g.,chickenpox and fever blisters) and are characterized by latent andrecurrent infections. HSV-2 is the leading cause of genital ulcers,which can manifest as a cluster of small fluid-filled blisters thatrupture and form painful sores, taking several weeks to heal. Additionalsymptoms may include fever, general sick feeling, muscle aches, painfulurination, vaginal discharge, and enlarged, tender lymph nodes in thegroin area. Recurring outbreaks are likely. The virus can exist in nervecells for the life time of the infected subject and reactivate, formingskin ulcers, at irregular intervals. Even in the absence of actualulcers, the virus can be produced and spread from individual toindividual. It is presently incurable.

Genital herpes is the most prevalent sexually transmitted disease. Inthe United States, over 16% of the population, or about one out of sixpeople, is infected with HSV-2, with a disproportionate burden onwomen—approximately 20% of women and 12% of men—and onAfrican-Americans—about 40% of the population and nearly 50% ofAfrican-American women. (Morbidity and Mortality Weekly Report, 59:456-459, Apr. 23, 2010). Altogether, about 50 million people in the U.S.are infected, of which about 80% are unaware of their infection, but maystill be infectious. Elsewhere in the world, HSV-2 also attains epidemicproportions. A WHO team estimated that in 2003, 536 million peopleworld-wide were infected, and new infections were occurring at about 23million yearly (Looker et al., Bull World Health Organ. 86: 805-812,2008). Although prevalence varied by region, generally prevalenceincreased with age and was higher among women than among men. Inaddition, HSV-2 prevalence is higher in developing countries than indeveloped countries—with the exceptions of North America, which has ahigh HSV-2 prevalence, and south Asia, which has a relatively low HSV-2prevalence. The highest prevalence is found in Sub-Saharan Africa wherenearly 80% of women and 45% of men are infected with HSV-2. Otherregions, notably eastern Asia and south-east Asia, approach this level.In addition to sexual transmission, HSV-2 can be transmitted from awoman to a baby, typically at the time of delivery. Concomitant with theHSV-2 epidemic in the adult U.S. population, the incidence of neonatalinfection has also dramatically increased. About 1,800 cases of neonatalHSV infection occur yearly in the U.S., which is a higher number ofcases than neonatal HIV infection.

The health implications of HSV-2 infection are staggering. Although thevast majority of infected individuals are asymptomatic, virus can stillbe transmitted. Those with symptoms suffer painful sores on theirgenitals and anal region and often flu-like symptoms such as fever andswollen glands. Unfortunately, those with a first outbreak of HSV-2 arelikely to have several additional outbreaks (typically four or five)within the first year alone. Regardless of the severity of symptoms,knowledge of infection often causes stress and can negatively impactquality of life (Rosenthal, et al., Sex Transm Infect. 82: 154, 2006;Crosby et al Sex Health, 5:279-283, 2008). In neonates infected withHSV-2, neonatal encephalitis from HSV infection has a mortality of >15%even with treatment, and the neurological morbidity among HSV-2 infectedinfants is an additional 30-50% of surviving cases Coupled with the highprevalence of HSV-2, there is a stark realization that HSV-2 infectionsubstantially increases the risk for HIV-1 acquisition and transmission.Data from Africa show that HSV-2 infection can increase the risk for HIVtransmission by as much as seven-fold and that up to one-half of newlyacquired HIV cases are directly attributed to HSV-2 infection. Overall,the relative risk of HIV acquisition increases more than two-fold inHSV-2-infected individuals. The synergistic effect on HIV acquisition isgreater for HSV-2 than for any other sexually transmitted infection,underscoring the need for an effective public health strategy capable ofminimizing the effects of the current HSV-2 epidemic.

The increasing prevalence of HSV-2 in the adult and pediatricpopulations persists despite the widespread use of pharmacologicalintervention. Antiviral medication, such as acyclovir, given at highdoses early in infection can reduce HSV transmission, but this does notprevent latent infection of the neuronal ganglion. Antiviral therapy hasmany drawbacks, including as side effects nausea, vomiting, rashes, anddecreased kidney function, and should be used with caution because theycan be teratogenic as well as be toxic to developing embryos.Furthermore, continuous suppressive administration with valcyclovirreduced HSV transmission by less than 50% despite early intervention.Even if this level of effect were acceptable, the approach isimpractical considering the high cost and that 80% of those infected areunaware of their status. Alternatives to antiviral drugs, such astopical microbicides are unproven clinically, and physical barriers(e.g., condoms) have marginal “real-world” efficacy. For these reasons,vaccination is essential for combating and diminishing the health impactof HSV-2 infection.

The first vaccine for HSV was developed in the 1920s, and since then, avariety of vaccine approaches have been tried—all to no avail. Theconventional, time-honored types of vaccines including whole,inactivated virus, attenuated live virus, modified live virus, and cellculture-derived subunits were largely unsuccessful or had low efficacy(Stanberry, Herpes 11 (Suppl 3) 161A-169A, 2004). With the advent ofrecombinant DNA technology, recombinant subunit vaccines have beendeveloped. These vaccines comprised one or two of the envelopeglycoproteins in combination with adjuvants. The glycoproteins wereattractive candidates mainly because they are the targets ofneutralizing antibodies and they are highly conserved among HSV-2strains. In the last decade, extensive clinical trials on two candidatevaccines, one developed by Chiron and the other by GlaxoSmithKline, wereboth halted due to insufficient efficacy. Chiron's vaccine comprisedtruncated forms of two HSV-2 glycoproteins, gD2 and gB2, in combinationwith the adjuvant MF59. The vaccine at best provided transientprotection against HSV-2 although high titers of antibodies to HSV-2were generated (Stanberry, ibid). GlaxoSmithKline (GSK) developed andtested a similar vaccine; however it contained only a singleglycoprotein, gD2, and alum and MPL as adjuvants. Following eight yearsof studies and clinical trials, GSK pronounced it as a failure inOctober 2010. The vaccine was unsuccessful in preventing infection inseronegative women, the only group in early clinical trials that hadseemed to benefit.

SUMMARY

In one embodiment of the disclosure, an immunogenic fragment of an HSV-2polypeptide is provided selected from the group consisting of: (a) animmunogenic fragment of UL19 polypeptide lacking at least 75% of aminoacids 1-450 of SEQ ID NO: 4 and lacking at least 75% of amino acids of1055-1374 of SEQ ID NO: 4; (b) the sequence set out in SEQ ID NO: 12;(c) an immunogenic variant of (a) or (b) that retains at least 85% aminoacid identity over at least 15 contiguous amino acids; (d) animmunogenic fragment of (a) or (b); and (e) a chimeric fusion of (a),(b), (c) or (d). In another embodiment an isolated polynucleotideencoding the aforementioned polypeptide is provided.

Pharmaceutical compositions are also provided by the instant disclosure.In one embodiment, an immunogenic, pharmaceutical composition isprovided comprising: (i) an immunogenic fragment of an HSV-2 polypeptideselected from the group consisting of: (a) an immunogenic fragment ofUL19 polypeptide lacking at least 75% of amino acids 1-450 of SEQ ID NO:4 and lacking at least 75% of amino acids of 1055-1374 of SEQ ID NO: 4;(b) the sequence set out in SEQ ID NO: 12; (c) an immunogenic variant of(a) or (b) that retains at least 85% amino acid identity over at least15 contiguous amino acids; (d) an immunogenic fragment of (a) or (b);and (e) a chimeric fusion of (a), (b), (c) or (d); (ii) optionally, anagent that activates innate immunity; and (iii) a pharmaceuticallyacceptable carrier.

In another embodiment, the aforementioned composition is provided whichfurther comprises UL25 or an immunogenic fragment thereof. In stillanother embodiment, the composition further comprises gD2 or animmunogenic fragment thereof.

In still another embodiment of the instant disclosure, theaforementioned composition is provided wherein the agent is an adjuvant.In one embodiment, the adjuvant is GLA. In another embodiment, the GLAis in the form of an oil-in-water emulsion or an aqueous form. Incertain embodiments, the oil-in-water emulsion comprises squalene.

In yet another embodiment of the disclosure, a method for treating anHSV-2 infection in a subject is provided comprising administering anaforementioned composition to the subject. In a another embodiment, amethod of generating an immune response in a subject comprisingadministering an aforementioned composition to the subject is provided.In still another embodiment, a method for immunizing a subject againstHSV-2 comprising administering an aforementioned composition to thesubject is provided. According to various embodiments of the disclosure,an aforementioned method is provided wherein the administration route isintradermal, mucosal, intramuscular, subcutaneous, sublingual, rectal,or vaginal. In still another embodiment, an aforementioned method isprovided further comprising administering a second, third or fourthcomposition according to any one of claims 3-8 to the subject.

The claimed invention is directed to compositions and methods useful inpreventing or treating HSV-2 (herpes simplex virus 2) infections insubjects, preferably humans, in one embodiment the human is female,while in another embodiment the human is male. The compositions comprise(i) an envelope glycoprotein of HSV-2 or an immunogenic fragment of theHSV-2 envelope glycoprotein, (ii) an HSV-2 structural protein orimmunogenic fragment of the HSV-2 structural protein, wherein thestructural protein is not one of the envelope glycoproteins, (iii) anagent that activates innate immunity in a subject and (iv) apharmaceutically acceptable carrier. In certain embodiments, theenvelope glycoprotein is gD2 and the composition has either gD2 or in analternative embodiment, an immunogenic fragment derived from gD2. Insome embodiments, the structural protein is one or more of UL47, ICP0,ICP4, ICP47, UL5, UL8, UL15, UL19, UL25, UL30, UL32, UL46, UL39 (ICP10),UL7, UL40, UL54 and UL26 and if immunogenic fragments are present, theyare derived from UL47, ICP0, ICP4, ICP47, UL5, UL8, UL15, UL19, UL25,UL30, UL32, UL46, UL39 (ICP10), UL7, UL40, UL54 and/or UL26. It isunderstood that the exact sequence of a protein may vary from oneherpesvirus to another, and thus all references to an HSV-2 proteinencompasses any such protein obtainable from any naturally occurringHSV-2. In other embodiments, both UL19 and UL25, or fragments from UL19(e.g. SEQ ID NO. 12, a type of Upper Domain Fragment) and UL25, or amixture of whole protein and fragments are present, e.g. a mixture offull length UL25 and a fragment of UL19, e.g., SEQ ID NO. 12, optionallywith UL47 or a fragment thereof. At times, the agent that activatesinnate immunity is an adjuvant. In particular the adjuvant can be GLA oranother MALA adjuvant. In one embodiment the immunogenic, pharmaceuticalcomposition comprises gD2, GLA or another MALA adjuvant, and two orthree antigens selected from full length or fragments of UL25, UL19, andUL47, and a pharmaceutically acceptable carrier. In related embodiments,the immunogenic, pharmaceutical composition comprises a MALA adjuvant,preferably GLA having the structural formula of FIG. 1, gD2, UL25, UL19Upper Domain Fragment, and a pharmaceutically acceptable carrier;optionally such a composition further comprises one or more additionalHSV-2 structural proteins, or fragments thereof.

In some embodiments, the compositions comprise an antigenic portion ofan envelope glycoprotein of HSV-2 and a pharmaceutically acceptablecarrier. The terms ‘immunogenic fragment” and “immunological fragment”and “antigenic portion” are used interchangeably herein to designatefragments or portions of proteins that elicit an antibody response or acellular cytotoxic response that retains specificity for(cross-reactivity with) the full length protein. In certain embodiments,the antigenic portion binds to neutralizing antibodies. In certainembodiments, the antigenic portion is from gD2 or gB2, and in otherembodiments, the antigenic portion, whether from gD2, gB2 or anotherenvelope glycoprotein, comprises at least part and optionally all of theleader sequence. In any of the embodiments, the antigenic portioncomprises two or more linear epitopes or comprises two or morediscontinuous epitopes from the envelope glycoprotein. In any of theembodiments, the composition further comprises an agent that activatesinnate immunity. The agent may be an adjuvant, such as GLA as disclosedin, for example, US Publication No. 2009/0181078.

The methods can be used to treat an HSV-2 infection or to generate animmune response, which may prevent or ameliorate an HSV-2 infection.Suitable subjects for the methods include those who are seropositive forHSV-2 as well as those who are seronegative for HSV-2. In the methods,one of the compositions described herein is administered to a subject.

Some exemplary statements of the present invention are set forth asfollows, using the designation (xy) where each of x and y denote aletter, the designation denoting an embodiment, or group of embodimentswhen more than one (xy) is identified within an embodiment. (AA) Animmunogenic, pharmaceutical composition comprising (i) an envelopeglycoprotein of HSV-2, or an immunological fragment thereof; (ii) astructural protein of HSV-2 other than an envelope glycoprotein ofHSV-2, or an immunological fragment thereof; (iii) an agent thatactivates innate immunity; and (iv) a pharmaceutically acceptablecarrier. (AB) Composition (AA) wherein the envelope glycoprotein ofHSV-2 is gD2, and the composition comprises gD2. (AC) Composition (AA)wherein the composition comprises an immunological fragment of gD2. (AD)A composition of any one or more of (AA), (AB) and (AC), wherein thestructural protein of HSV-2 is one or more proteins selected from thegroup consisting of UL47, ICP0, UL25, UL46, UL39, UL7, and UL26. (AE)Composition (AA) wherein the structural protein of HSV-2 is UL19. (AF)The composition of (AB) wherein the structural protein of HSV-2 is UL19.(AG) Composition (AA) wherein the structural protein of HSV-2 is animmunological fragment of UL19, e.g., SEQ ID NO. 12. (AH) Composition(AB) wherein the structural protein of HSV-2 is an immunologicalfragment thereof UL47. (AI) Composition (AA) wherein the structuralprotein of HSV-2 is UL25. (AJ) Composition (AB) wherein the structuralprotein of HSV-2 is UL25. (AK) Composition (AA) wherein the structuralprotein of HSV-2 is an immunological fragment of UL25. (AL) Composition(AB) wherein the structural protein of HSV-2 is ICP0. (AM) Composition(AA) wherein the structural protein of HSV-2 is UL47. (AN) Composition(AB) wherein the structural protein of HSV-2 is a fragment of UL47. (AO)Composition (AA) wherein the structural protein of HSV-2 other than anenvelope glycoprotein of HSV-2 is UL47, and is an immunological fragmentthereof. (AP) Composition (AB) wherein the structural protein of HSV-2other than an envelope glycoprotein of HSV-2 is UL47, and is animmunological fragment thereof. (AQ) A composition of any one or more of(AA), (AB), (AC), (AD), (AE), (AF), (AG), (AH), (AI), (AJ), (AK), (AL),(AM), (AN), (AO), (AP) further comprising a second structural protein ofHSV-2 other than an envelope glycoprotein of HSV-2, or an immunologicalfragment thereof. (AR) Composition (AQ) wherein the second structuralprotein of HSV-2 other than an envelope glycoprotein of HSV-2 isselected from the group consisting of UL19, UL25 and UL47, where thesecond structural protein is non-identical to the structural protein.(AS) Composition (AR) comprising the second structural protein. (AT)Composition (AR) comprising an immunological fragment of the secondstructural protein. (AU) A composition of any one or more of (AE), (AF),(AG) and/or (AH) further comprising UL25. (AV) A composition of any oneor more of (AE), (AF), (AG) and/or (AH) further comprising animmunological fragment of UL25. (AW) A composition of any one or more of(AE), (AF), (AG) and/or (AH) further comprising UL47. (AX) A compositionof any one or more of (AE), (AF), (AG) and/or (AH) further comprising animmunological fragment of UL47. (AY) A composition of any one or more of(AI), (AJ), (AK) and/or (AL) further comprising UL19. (AZ) A compositionof any one or more of (AI), (AJ), (AK) and/or (AL) further comprising animmunological fragment of UL19, e.g., SEQ ID NO 12. (BA) A compositionof any one or more of (AI), (AJ), (AK) and/or (AL) further comprisingUL47. (BB) A composition of any one or more of (AI), (AJ), (AK) and/or(AL) further comprising an immunological fragment of UL47. (BC) Acomposition of any one or more of (AM), (AN), (AO) and/or (AP) furthercomprising UL19. (BD) A composition of any one or more of (AM), (AN),(AO) and/or (AP) further comprising an immunological fragment of UL19.(BE) A composition of any one or more of (AM), (AN), (AO) and/or (AP)further comprising UL25. (BF) A composition of any one or more of (AM),(AN), (AO) and/or (AP) further comprising an immunological fragment ofUL25. (BG) A composition of any one or more of (AA), (AB), (AC), (AD),(AE), (AF), (AG), (AH), (AI), (AJ), (AK), (AL), (AM), (AN), (AO), (AP),(AQ), (AR), (AS), (AT), (AU), (AV), (AW), (AX), (AY), (AZ), (BA), (BB),(BC), (BD), (BE), and (BF) wherein the agent is an adjuvant. (BH) Acomposition selected from (BG) wherein the adjuvant is GLA or anotherMALA adjuvant, and each and every one of the options in (BG) isindependently selected as a distinct embodiment of the presentinvention. (BI) Composition (AA) comprising gD2; UL25; UL19; GLA oranother MALA adjuvant; and a pharmaceutically acceptable carrier. (BJ)Composition (AA) comprising gD2, UL25 and an immunological fragment ofUL19. (BK) Composition (AA) comprising gD2, UL19, and an immunologicalfragment of UL25. (BL) A composition of any one or more of (BI), (BJ)and (BK) further comprising UL47. (BM) A composition of any one or moreof (BI), (BJ) and (BK) further comprising an immunological fragment ofUL47. (BN) A method for treating an HSV-2 infection in a subject,comprising administering the composition of any one or more of (AA),(AB), (AC), (AD), (AE), (AF), (AG), (AH), (AI), (AJ), (AK), (AL), (AM),(AN), (AO), (AP), (AQ), (AR), (AS), (AT), (AU), (AV), (AW), (AX), (AY),(AZ), (BA), (BB), (BC), (BD), (BE), (BF), (BG), (BH), (BI), (BJ), (BK),(BL), and (BM) to the subject. (BO) A method for generating an immuneresponse to HSV-2 in a subject, comprising administering the compositionof any one or more of (AA), (AB), (AC), (AD), (AE), (AF), (AG), (AH),(AI), (AJ), (AK), (AL), (AM), (AN), (AO), (AP), (AQ), (AR), (AS), (AT),(AU), (AV), (AW), (AX), (AY), (AZ), (BA), (BB), (BC), (BD), (BE), (BF),(BG), (BH), (BI), (BJ), (BK), (BL), (BM), and (BN) to the subject. (BQ)Method (BO) wherein the subject is seropositive for HSV-2 andseropositive for HSV-1. (BR) Method (BO) wherein the subject isseropositive for HSV-2 and seronegative for HSV-1.

In one embodiment there is provided a composition comprising an envelopeglycoprotein of HSV-2 or an immunological fragment thereof; twostructural proteins of HSV-2 other than an envelope glycoprotein ofHSV-2, or an immunological fragment thereof; an agent that activatesinnate immunity; and a pharmaceutically acceptable carrier. Exemplary isa composition that comprises gD2, UL25, and SEQ ID NO. 12 (a fragment ofUL19) and a monophosphoryl lipid A (MALA) adjuvant, e.g., GLA. Inaddition to gD2-specific antibody responses, vaccination with thiscomposition may elicit robust HSV-2 antigen-specific CD4 and CD8effector and memory T cells that respond to subsequent infection withlive virus. Notably, prophylactic immunization with this composition maylargely or completely protect against lethal intravaginal HSV-2infection in C57BL/6 mice, with sterilizing immunity in both the genitalmucosa and dorsal root ganglia. This composition may expand both CD4 andCD8 T cells induced by previous infection with an attenuated strain ofHSV-2. Consistent with this, when applied as a therapy for recurrentHSV-2 lesions in guinea pigs, this composition may reduce the frequencyof recurrent lesions.

Kits are also provided. In some kits, there is a vial comprising thepharmaceutical composition comprising an antigenic portion of an HSV-2envelope glycoprotein and a pharmaceutically acceptable carrier.

These and other aspects and embodiments of the present invention willbecome evident upon reference to the following detailed description andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B present a drawing of GLA (the adjuvant used in the Examples)and a schematic of an exemplary oil droplet with surfactantsphosphatidycholine and Pluronic F68.

FIG. 2 shows gD2-specific CD4 T cell responses. Data were obtained afterBalb/c mice (4/group) were immunized twice i.m. at a 28 day intervalwith a bivalent vaccine comprised of varying levels of recombinantprotein and GLA, as indicated. The graphs are results of flow cytometryanalyses for intracellular production of IL-2, TNF-α, and IFN-γ.

FIG. 3 shows splenic CD8 T cell responses to OVA257 peptide analyzed onD25 post-prime (D4 post-Boost); recombinant OVA=5 μg; SE=2%; lentivirusdelivered s.c.; recombinant OVA delivered i.m.

FIG. 4 is a graph showing percent cytokine positive CD8 T cells measured4 days after a boost. Priming took place on day 0 and boosting on day21. Column HAL d0 HBSS, d21, PBS; HA2, d0, LV-OVA, d21, PBS; HA3, d0LV-OVA, d21 LV-OVA; HA4, d0 LV-OVA, d21 20 μg GLA-SE; HA5, d0 LV-OVA,d21 OVA+SE; HA6, d0 LV-OVA, d21 OVA+20 μg GLA-SE; HA7, d0 LV-OVA, d21, 4μg OVA+GLA-SE; HA8, d0 LV-OVA, d21 OVA+0.8 μg GLA-SE.

FIGS. 5A-B show data obtained after groups of C57BL/6 mice (5/group)were immunized via a prime/boost immunization regimen (d0 prime/d21boost) with either 5 μg of recombinant gD, UL19, or UL25 protein incombination with 5 μg of GLA-SE. Splenic CD4 T cell responses weremeasured on day 4 post-boost by intracellular staining for IFN-γ, TNF-α,and IL-2 after ex vivo re-stimulation with 15-mer peptides previouslyidentified as containing CD4 epitopes for the corresponding recombinantprotein immunogen. A) Representative ICS dot plot of the CD4 T cellresponse to each 15-mer peptide indicated in mice immunized with thecorresponding recombinant protein immunogen. B) Percent cytokinepositive CD4 T cells are depicted for each group.

FIGS. 6 A-B show data obtained after a group of five C57BL/6 mice wereimmunized via a prime/boost regimen (d0 prime/d21 boost) withrecombinant gD, UL19, and UL25 proteins delivered in combination andformulated on an equimolar basis (0.8, 3.3, and 1.4 μg of protein,respectively) in combination with 5.5 μg of GLA-SE. Splenic CD4 T cellresponses were measured on day 4 post-boost by intracellular stainingfor IFN-γ, TNF-α, and IL-12 after ex vivo restimulation with 15-merpeptides previously identified as containing CD4 T cell epitopes foreach recombinant protein immunogen. An individual peptide which lacks aCD4 T cell epitope from each peptide library was included as a negativecontrol. A) percent cytokine positive CD4 T cells are depicted for eachgroup. B) Serum endpoint titers (defined as the reciprocal of thehighest serum dilution that is >2 times background) for antigen-specificantibodies of the IgG1 subclass for each recombinant protein immunogenwithin the trivalent vaccine.

FIGS. 7 A-B show data obtained when groups of C57BL/6 mice (5/group)were immunized via a prime (d0) or prime boost (d0 prime/d21 boost)immunization regimen with 5 μg recombinant UL19 protein delivered incombination with 5 μg of GLA-SE. Splenic CD4 T cell responses weremeasured on day 4 or day 10 post-last immunization by ICS for IFN-γ,TNF-α, and IL-12 after ex vivo re-stimulation with 15-mer peptidespreviously identified as containing CD4 T cell epitopes for UL19. A)Representative ICS dot plots of the CD4 T cell response to UL19 15-merpeptide 297 indicated in mice immunized with the correspondingrecombinant protein immunogen. Percent cytokine positive DC4 T cells aredepicted for each group. B) Percent cytokine positive CD4 T cellsresponding to UL19 15-mer 250 or 297 are depicted for each group.

FIGS. 8A-B show data obtained when groups of C57BL/6 mice (5/group) wereimmunized via a prime (d0) or prime boost (d0 prime/d21 boost)immunization regimen with 5 μg recombinant UL19 protein delivered aloneor in combination with 5 μg of SE or GLA-SE. Splenic CD4 T cellresponses were measured on day 5 or day 10 post-last immunization by ICSfor IFN-γ, TNF-α, and IL-12 after ex vivo re-stimulation with 15-merpeptides previously identified as containing CD4 T cell epitopes forUL19. A) Representative ICS dot plots of the CD4 T cell response to UL1915-mer peptide 297 indicated in mice immunized with the correspondingrecombinant protein immunogen. Percent cytokine positive CD4 T cells aredepicted for each group. B) Percent cytokine positive CD4 T cellsresponding to UL19 15-mer 250 or 297 are depicted for each group.

FIGS. 9A-C show data obtained when groups of C57BL/6 mice (5/group) wereimmunized via a prime boost (d0 prime/d21 boost) immunization regimenwith recombinant proteins formulated on either an equimolar or anequimass basis. Total protein delivered was either 5 μg or 15 μg.Splenic CD4 T cell responses were measured on day 5 post-lastimmunization by intracellular staining for IFN-γ, TNF-α, and IL-12 afterex vivo re-stimulation with 15-mer peptides previously identified ascontaining CD4 T cell epitopes. A) Percent cytokine positive CD4 T cellsresponding to gD peptides are depicted. B) Percent cytokine positive CD4T cells responding to UL19 peptides are depicted. C) Percent cytokinepositive CD4 T cells responding to UL25 peptides are depicted.

FIG. 10 shows data obtained when groups of BALB/c mice (5/group) wereimmunized via a prime/boost immunization regimen (d0 prime/d21 boost)with 4 μg of recombinant gD protein in combination with either 4 μg ofGLA-SE, SE alone, or PBS vehicle, delivered intramuscularly in 100 μl(50 μl per leg). HSV-2 gD2-specific antibodies of the IgG, IgG1, andIgG2a isotypes were measured by ELISA.

FIG. 11 shows data obtained when groups of five C57BL/6 mice were givena single intramuscular immunization of trivalent vaccine consisting of 5μg each of recombinant gD2, UL19ud, and UL25 in combination with 5 μgGLA-SE or control vaccine articles. Antigen-specific splenic CD4 and CD8T cell responses were measured on day 6 post-immunization byIntracellular Cytokine Staining (ICS) for IFN-γ, TNF-α, and IL-2 afterex-vivo re-stimulation of splenocyte cultures for 5 hours with gD2,UL19, or UL25 peptides. A) Frequency and cytokine phenotype of CD4 Tcells responding to peptides from gD2, UL19ud, or UL25. B) Frequency andcytokine phenotype of CD8 T cells responding to UL19 peptides. C)Frequency of CD8 T cells responding to UL19 peptides in mice that wereimmunized 4 weeks earlier with trivalent vaccine with GLA-SE andchallenged subcutaneously with attenuated HSV-2 thymidinekinase-deficient (TK-) virus.

FIG. 12 shows data obtained when groups of ten C57BL/6 mice were giventwo intramuscular immunizations, separated by 28 days, of bivalentvaccine consisting of 5 μg each of recombinant gD2 and UL19ud incombination with either 5 μg GLA-SE or 5% dextrose vehicle. Miceimmunized with 5 μg GLA-SE alone served as negative controls. 22 daysafter the second immunization, mice were treated with depotmedroxyprogesterone acetate and then challenged six days later with a50×LD₅₀ dose of wild-type HSV-2 intravaginally. Mice monitored daily forformation of genital lesions and survival. On days 1, 3, and 5 postinfection, vaginal swabs were collected for quantitation of HSV-2 DNA byPCR. Approximately 2 months post infection, the dorsal root ganglia wereharvested from surviving mice and latent HSV-2 DNA was quantified byPCR. As depicted in FIG. 12, panel A, mice immunized with gD2 and UL19udwith GLA-SE has dramatically reduced lesion formation and increasedsurvival compared to mice immunized with either gD2 and UL19ud alone orGLA-SE alone. Likewise, as depicted in FIG. 12, panel B, 9 out of 10mice immunized with gD2 and UL19ud with GLA-SE had no detectable HSV-2DNA by day 5, whereas mice in either control group showed sustainedlevels of HSV-2 in the vagina through day 5. As depicted in FIG. 12,panel C, though there were three survivors in the GLA-SE only group, 2out of 3 of these mice showed significant levels of latent HSV-2 in thedorsal root ganglia, mice immunized with gD2 and UL19ud with GLA-SEshowed little to no detectable HSV-2 in the ganglia.

FIG. 13 shows data obtained when C57BL/6 mice (5/group) were infectedsubcutaneously with a sublethal dose of attenuated HSV-2 thymidinekinase-deficient (TK-) virus, then immunized 28 days later with atrivalent vaccine consisting of 5 μg each of recombinant gD2, UL19ud,and UL25 in combination with 5 μg GLA-SE or 5% dextrose vehicle. Controlgroups included infected mice treated with GLA-SE alone or vehiclealone, as well as naïve mice treated with vehicle alone. Six days postimmunization, UL19-specific CD8 (upper panel) and CD4 (lower panel) Tcell responses were measured by ICS after stimulation with UL19peptides.

FIG. 14 shows data obtained when guinea pigs (7/group) were infectedintravaginally with a sublethal dose of HSV-2 strain 333 virus and thentreated on days 13 and 27 post infection with trivalent vaccineconsisting of 5 μg each of recombinant gD2, UL19ud, and UL25 incombination with 5 μg GLA-SE. Infected guinea pigs treated with GLA-SEalone served as negative controls. Animals were monitored daily forvaginal lesions and scores of 0-4 were assigned for each lesion day.Daily lesions scores in each group were averaged and plotted versustime.

FIG. 15 shows data obtained when groups of ten C57BL/6 mice were giventwo intramuscular immunizations, separated by 28 days, of trivalentvaccine consisting of 5 μg each of recombinant gD2, UL19ud (see SEQ IDNO:12) and UL25 in combination with either 5 μg GLA-SE or 5% dextrosevehicle. Mice immunized with 5 μg GLA-SE alone served as negativecontrols. An additional control group consisted of mice immunized with 5μg GLA-SE and 1 milligram per ml of aciclovir (ACV) in the drinkingwater starting 24 hours after challenge. Twenty-two days after thesecond immunization, mice were treated with depot medroxyprogesteroneacetate and then challenged six days later with a 50×LD₅₀ dose ofwild-type HSV-2 intravaginally. Mice were monitored daily for formationof genital lesions (panel A) and survival (panel B).

FIG. 16: shows vaginal HSV-2 DNA levels in mice immunized with trivalentgD2, UL19ud (SEQ ID NO:12) and UL25 vaccine (see FIG. 15 for descriptionof groups of mice). Vaginal swabs were collected on days 1, 3, and 5post infection, for quantitation of HSV-2 DNA by PCR.

DETAILED DESCRIPTION

The present disclosure provides immunogenic, pharmaceutical compositionsand methods for treatment of or for prevention of herpes simplex virusinfections, including HSV-1 and HSV-2 infections. The compositionscomprise immunogenic HSV-2 viral proteins or immunogenic portions of theviral proteins, such as fragments or peptides, and at least one agentthat activates the innate immune system, preferably a TLR4 agonist, forexample, a MALA adjuvant as described herein. The viral proteins (andfragments and peptides) comprise at least one envelope glycoprotein andat least one, two, three or four structural proteins other than anenvelope glycoprotein. Alternatively, the viral proteins (and fragmentsand peptides) comprise at least one antigenic epitope and may comprisepart of or all of a leader peptide of an envelope protein. Immunogenicfragments may be used. Some specific agents useful in the compositionsinclude adjuvants, substances that enhance the immune response to anantigen. The proteins and fragments are typically produced by arecombinant technology in which the protein(s) or fragment(s) areexpressed in cultured cells. Peptides can also be chemicallysynthesized.

A. HSV-2 Protein as a Component of a Vaccine

HSV-2 (herpes simplex virus type 2) is an enveloped virus. Its genomeexpresses over 75 different proteins. Many of the proteins arestructural and are used to form the capsid and tegument, while someothers are part of the envelope. Major capsid proteins include thoseexpressed from open reading frames (protein names are in parentheses ifthe common name differs from the ORF name) UL6, UL18 (VP23), UL19 (VP5),UL35 (VP26) and UL38; major tegument proteins include UL7, UL11, UL13,UL14, UL16, UL17, UL21, UL25, UL36, UL37, UL41, UL46 (VP11/12), UL47(VP13/14), UL48 (VP16), UL49, UL51, and US11; major envelope proteinsinclude UL1 (glycoprotein L (gL)), UL10 (gM), UL20, UL22 (gH), UL27(gB), UL43, UL44 (gC), UL49A (gN), UL53 (gK), US4 (gG), US5, (gJ), US6(gD), US7 (gI), and US8 (gE). (Other protein names may have been used inthe literature.) An exemplary HSV-2 genome sequence is found in GenBankAccession No. NC 001798.1 (update date 23 Apr. 2010, 2:16 pm, accessed10 Jan. 2011; incorporated in its entirety). It is understood that thecommonly used protein names may be different from the gene names, e.g.UL19 encodes VP5, but reference to the gene name herein is the same as areference to the encoded protein. It is also understood that the exactsequence of a protein may vary from one herpesvirus to another, and thusall references to an HSV-2 protein (structural or envelope ornon-envelope) encompass any such protein obtainable from any naturallyoccurring HSV-2. A number of sequences are already known and depositedin databases. Nucleic acid encoding an HSV-2 protein with an alternativesequence can be readily isolated or amplified from one or more HSV-2(e.g. a deposited HSV-2 or a clinical isolate) with appropriateoligonucleotide probes or primers (e.g. that specifically hybridize to areference sequence under stringent conditions). Within such a group ofnucleic acids that encode an HSV-2 protein, e.g. an UL protein, onenucleic acid of the group will hybridize to the complement of anothernucleic acid within the group, under stringent conditions.

The term “stringent conditions” refers to conditions under which a probewill hybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and Northernhybridizations are sequence-dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids is found in Tijssen, Laboratory Techniques in Biochemistryand Molecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 in “Overview of principles of hybridization and the strategyof nucleic acid probe assays”, Elsevier (New York, 1993). In certainembodiments, highly stringent hybridization and wash conditions areabout 5° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. In certainembodiments, very stringent conditions are equal to the T_(m) for aparticular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids that have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes (see Sambrook et al. for adescription of SSC buffer). A high stringency wash can be preceded by alow stringency wash to remove background probe signal. An example ofmedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example of low stringency wash fora duplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for15 minutes. In general, a signal to noise ratio of 2× (or higher) thanthat observed for an unrelated probe in the particular hybridizationassay indicates detection of a specific hybridization.

Because one or more envelope proteins is involved in viral entry intohost cells, antibodies to envelope proteins can neutralize the virus,that is prevent infection or re-infection by the virus. Without wishingto be held to a mechanistic theory, eliciting antibodies to one or moreof those envelope proteins necessary for cellular entry is one way toobtain neutralizing antibodies. Vaccines comprising whole virus,typically inactivated virus, naturally present envelope proteins toimmune cells. For a vaccine comprising individual viral proteins, onestrategy to obtaining a neutralizing antibody response is to include oneor more envelope proteins or immunogenic protein fragments orimmunogenic peptides or some combination of these in a vaccine.

HSV-2 encodes 14 or more envelope-associated proteins, at least some ofwhich are involved with cellular entry, including but not limited to gB,gD, gH, and gL. gD appears to bind specifically to an HSV-2 receptor oncells, and gB, along with the heterodimer gH/gL, appears to mediatemembrane fusion. Thus, these four envelope glycoproteins are excellentchoices as immunogens for inclusion in a vaccine because antibodieselicited to these envelope glycoproteins may include neutralizingantibodies. Alternatively, or in addition, envelope glycoproteinsinvolved in virus shedding are also candidates as immunogens forinclusion in a vaccine.

Most of the structural proteins of HSV-2 other than envelope proteinsare found in the capsid and the tegument. The tegument occupies thespace between the capsid and the envelope. There are approximately 20viral proteins found in the tegument. Tegument proteins are importantfor a variety of viral functions, including immune modulation, viralassembly and final egress. Capsid proteins form a structure thatsurrounds the nucleic acid genome of the virion. VP5, the product ofUL19 is the major capsid protein. A cellular response is often elicitedto structural proteins and to a variety of HSV proteins (Hosken et al.,J Virol 80:5509-55515, 2006). Generally, the cellular response involvesboth CD4 and CD8 T cells, cell types that play a role in combating HSVinfections.

The immunogenic, pharmaceutical composition (e.g., a vaccine) disclosedherein comprises as immunogens two or more structural proteins, one ofwhich is an envelope glycoprotein and another of which is other than anenvelope glycoprotein. Although any of the structural proteins can beused, the choice may be guided by ease of production, ability toformulate into a pharmaceutical composition, information on proteinstructure, and high expression levels. Because T cell responses aretypically MHC-restricted, a vaccine generally contains proteins orpeptides that are responded to by the highest number of MHC types, andit may also contain multiple proteins or peptides in order to increasenumber of individuals that will respond.

Immunogenic pharmaceutical compositions are preferably sterile, free orsubstantially free of other viral contaminants, and free orsubstantially free of pyrogenic substances such as LPS. Suchcompositions are for use as vaccines.

The envelope and non-envelope structural proteins for use in a vaccineas immunogens are typically full-length, but can also be a precursorprotein, fragment, or part of a fusion protein. A full-length proteinrefers to a mature protein; for example, in the case of an envelopeprotein, a mature protein is the form found in the envelope (e.g.,lacking a leader peptide). A precursor protein (pre-protein) is thenascent, translated protein before any processing occurs or apartially-processed protein. As part of a fusion protein, the HSV-2protein may be present as a precursor or full-length protein or aprotein fragment. A fragment of a protein should be immunogenic,containing one or more epitopes that elicit an immune response.

In some embodiments, the immunogenic, pharmaceutical composition (e.g.,a vaccine) disclosed herein comprises as immunogens (i) an α group geneproduct of HSV-2, or an immunological fragment thereof; and/or (ii) a β1group gene product of HSV-2, or an immunological fragment thereof;and/or (iii) a β2 group gene product of HSV-2, or an immunologicalfragment thereof; and/or (iv) a γ1 group gene product of HSV-2, or animmunological fragment thereof; and/or (v) a γ2 group gene product ofHSV-2, or an immunological fragment thereof. The α, β1, β2, γ1, and γ2genes are well-known in the art. See, for example, Herpesviruses andTheir Replication in FUNDAMENTAL VIROLOGY, Chapter 29, 1986.

Thus, any use of the term “immunogen” herein refers to the entire groupof polypeptides that are: (a) full length antigen, (2) immunogenicfragments of the antigen, (3) immunogenic variants of the full lengthantigen or variants of an immunogenic fragment, (4) chimeric fusionsthereof comprising portions of a different polypeptide, and (5)conjugates thereof. In various embodiments, the envelope andnon-envelope structural proteins for use in a vaccine include apolypeptide comprising any of an immunogenic fragment thereof or avariant thereof capable of inducing an immune response specific for theprotein.

For example, immunogenic variants retain at least 90% amino acididentity over at least 10 contiguous amino acids of the antigen, or atleast 85% amino acid identity over at least 15 contiguous amino acids ofthe antigen (e.g. an envelope protein or non-envelope structuralprotein). Other examples include at least 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%. 98%, or 99% identity over at least 50contiguous amino acids of the antigen, or over at least 100 contiguousamino acids of the antigen. In one embodiment, an immunogenic varianthas at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.98%, or 99% identity over the full length of a particular antigen. Insome embodiments, the variant is a naturally occurring variant.

As another example, immunogenic fragments, and variants thereof,comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48 or 50 contiguous aminoacids of the antigen. The immunogenic fragment may comprise any numberof contiguous amino acids between the aforementioned such that, forexample, an immunogenic fragment is between about 6-10, 10-15, 15-20,20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or morecontiguous amino acids of an immunogenic polypeptide.

Short fragments, often called peptides, are chosen to complex with MHCmolecules for binding to T cell receptors and are generally up to about30 amino acids long, or up to about 25 amino acids long, or up to about20 amino acids long, or up to about 15 amino acids long, up to about 12amino acids long, up to about 9 amino acids long, up to about 8 aminoacids long. In general, shorter peptides bind to or associate with MHCClass I molecules and longer peptides bind to or associate with MHCClass II molecules. Suitable peptides can be predicted using any of anumber of bioinformatic programs and tested using well-known methods.Short fragments, also called herein “peptides” are typically from 15-100amino acids long; longer fragments typically are from 100 amino acids upto full-length, although the length ranges for peptides (shortfragments) and longer fragments are not rigid.

As disclosed herein, suitable proteins include precursor proteins,mature proteins, fragments, fusion proteins and peptides. In thecompositions, the proteins may be present in the same form or as amixture of these forms. For example, an envelope glycoprotein may bepresent as a mature protein and a structural protein as a fragment or anenvelope glycoprotein may be present as a fragment and a structuralprotein as a fragment. For cellular production of the glycoprotein, asignal peptide may be part of the precursor protein. Signal peptidesinclude the glycoprotein D native sequence or others known in the art.It may also be desirable to use a protein without a transmembrane orintracellular region or both.

As discussed herein, one or more portions, also called fragments, of anenvelope glycoprotein are chosen for containing one or more epitopesthat bind to neutralizing antibodies. Portions containing epitopes maybe identified by an assay, such as inhibition of neutralizing antibodieson viral infection of cells. Briefly, overlapping portions of an HSV-2envelope glycoprotein are mixed with neutralizing antibodies (e.g.,serum from an infected animal or human), and the mixture added to HSV-2and a permissive cell line. If a portion has an epitope that binds tothe antibodies, the cell line will be infected with HSV-2. If theportion doesn't have an epitope, the cell line will not be infected.

Compositions that comprise at least one immunogenic fragment of animmunogenic HSV-2 polypeptide may be used as immunogens. In someembodiments, the immunogenic fragment is encoded by the recombinantexpression vectors described herein. The immunogenic fragment mayconsist of at least 6, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore contiguous amino acids of an immunogenic polypeptide. Theimmunogenic fragment may comprise any number of contiguous amino acidsbetween the aforementioned such that, for example, an immunogenicfragment is between about 6-10, 10-15, 15-20, 20-30, 30-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, or more contiguous amino acids of animmunogenic polypeptide. The immunogenic fragments may comprise asufficient number of contiguous amino acids that form a linear epitopeand/or may comprise a sufficient number of contiguous amino acids thatpermit the fragment to fold in the same (or sufficiently similar)three-dimensional conformation as the full-length polypeptide from whichthe fragment is derived to present a non-linear epitope or epitopes(also referred to in the art as conformational epitopes). Assays forassessing whether the immunogenic fragment folds into a conformationcomparable to the full-length polypeptide include, for example, theability of the protein to react with mono- or polyclonal antibodies thatare specific for native or unfolded epitopes, the retention of otherligand-binding functions, and the sensitivity or resistance of thepolypeptide fragment to digestion with proteases (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring HarborLaboratory Press, NY (2001)). Accordingly, by way of example, thethree-dimensional conformation of a polypeptide fragment is sufficientlysimilar to the full-length polypeptide when the capability to bind andthe level of binding of an antibody that specifically binds to thefull-length polypeptide is substantially the same for the fragment asfor the full-length polypeptide (i.e., the level of binding has beenretained to a statistically, clinically, and/or biologically sufficientdegree compared with the immunogenicity of the exemplary or wild-typefull-length antigen).

Fragments that are screened in an assay, such as that described above,are generally short. Generally, the length of a candidate fragment is upto about 40 amino acids long, or up to about 25 amino acids long, or upto about 20 amino acids long, or up to about 15 amino acids long, or upto about 12 amino acids long, or up to about 9 amino acids long, or upto about 8 amino acids long. Fragments used for screening are typicallyoverlapping. For example, a set of fragments might comprise 20 aminoacid long fragments that overlap by 16 amino acids (i.e., staggeredevery 4 amino acids). Typically, the overlapping sets start at theN-terminus of an unprocessed glycoprotein, i.e., contains a leadersequence, and ends at the C-terminal amino acid of the extracellulardomain.

Fragments that bind to neutralizing antibody are chosen and may be usedin a pharmaceutical composition as disclosed herein. The fragments maybe used “as-is” or engineered further or in combination with otherfragments. For fragments that are big enough and complex enough to beimmunogenic, they may be used in pharmaceutical compositions. Fragmentsless than about 1000 MW are unlikely to be immunogenic, althoughcomplexity can also play a role in whether a fragment is immunogenic.For example, homopolymers consisting of repeating units of a singleamino acid are poor immunogens regardless of their size, whereasco-polymers of 2 or 3 amino acids may be good immunogens. A co-polymerof glutamic acid and lysine needs to be at least about 30-40,000 MW tobe immunogenic. Amino acids with aromatic side chains increaseimmunogenicity, such that a fragment of only about 4000 MW thatcomprises tyrosine and phenylalanine may be immunogenic. Fragments thatare too short or not complex enough to be immunogenic may be conjugatedto a carrier protein, such as KLH (keyhole limpit hemocyanin),ovalbumim, bovine serum albumin, or other protein that is foreign to thesubject receiving the pharmaceutical composition, or the fragments maybe coupled together to create an immunogenic protein. Whether or not afragment is immunogenic may be determined in an animal. For example, thefragment may be administered to an animal in a prime-boost regimen, andantibodies to the fragment assayed in an e.g., ELISA using serum drawn7-10 days following the boost. A detectable signal indicates that thefragment is immunogenic. Higher signals are desirable. Other assays forimmunogenicity are well known to one of average skill.

In some embodiments, the fragments used in the compositions aresynthetic long peptides. “Synthetic long peptide” (SLP) refers to aprotein sequence manufactured ex vivo and having a length as short asabout 25 amino acids and as long as about 100 amino acids. An SLP shouldbe long enough to be taken up and processed by dendritic cells forpresentation on their cell surface with MHC class I or class IImolecules. SLPs are peptides derived from proteins against which animmune response is desired. In one embodiment, the immune response is aT cell response. The proteins may be known antigens or, in the case ofsome proteins, they may be candidate antigens.

An SLP comprises at least one CD4 epitope or at least one CD8 epitope orat least one CD4 and at least one CD8 epitope. A CD4 epitope refers toan amino acid sequence that binds to class II MHC and a CD8 epitoperefers to an amino acid sequence that binds to class I MHC. Epitopesequences are derived from the amino acid sequence of an immunogen; invivo, briefly, the immunogen is taken up or synthesized byantigen-processing cells (e.g., dendritic cells) and degraded intopeptides, which associate with MHC molecules and are presented on thecell surface as an MHC-peptide complex. Peptides complexed with MHCclass I molecules interact with the T cell antigen receptor and CD8 onCD8+ T cells, these peptides are called CD8 epitopes; peptides complexedwith MHC class II molecules interact with T cell antigen receptor andCD4 on CD4+ T cells, these peptides are called CD4 epitopes. ActivatedCD8+ T cells become cytotoxic T cells, which recognize and kill targetcells displaying the MHC class I-CD8 epitopes. Often, target cells areinfected or tumor cells. Activated CD4+ T cells become helper T cells,and depending on their subtype, help B cells to produce antibody oractivate natural killer cells, phagocytes and CD8+ T cells. Activationof both CD4+ T cells and CD8+ T cells contribute to a comprehensivecellular immune response.

As disclosed above, an SLP should be long enough to be taken up andprocessed by dendritic cells and presented on their cell surface withMHC molecules. Peptides complexed with MHC class 1 molecules aregenerally 8-11 amino acids in length, and peptides complexed with MHCclass II molecules are generally 13-17 amino acids in length, althoughlonger or shorter lengths are not uncommon. As such, an SLP willtypically be at least 25 amino acids long and as long as 100 amino acidslong (e.g., at least 30 aa, at least 35 aa, at least 40 aa, at least 45aa, at least 50 aa, at least 55 aa, at least 60 aa, at least 65 aa, atleast 70 aa, at least 75 aa, at least 80 aa, at least 85 aa, at least 90aa, at least 95 aa). The length of an SLP will generally be about 45 aaor about 50 aa in length.

Epitopes may have known sequence or unknown sequence. A plethora ofproteins have been mapped for CD4 and CD8 epitopes. For SLPs comprisingone or more of these epitopes, the length will typically be about 45 aa.Moreover, the epitope may be flanked by about 15 aa at the N-terminaland at the C-terminal sides. The flanking sequences are typically thesequences that flank the epitope sequence in the native protein. Asdiscussed above, an SLP may comprise more than one epitope, the multipleepitopes may be all CD4 or CD8 epitopes or a mixture of CD4 and CD8epitopes. Furthermore, the epitopes may overlap in sequence (see Example1 for some exemplary SLPs that comprise overlapping epitopes). The totalnumber of SLPs used may be such that all known CD4 and CD8 epitopes arerepresented.

SLPs may be synthesized by any of a variety of methods (see Corradin etal., Sci Translational Med 2:1, 2010 for a general discussion ofsynthesis methods). Automated peptide synthesizers are commerciallyavailable, and many companies provide synthesis services (e.g.,Abbiotec, American Peptide Company, AnaSpec, Bachem, Covance ResearchProducts, Invitrogen). Following synthesis, peptides are purified,typically by HPLC, although alternative purification methods such as ionexchange chromatography and gel filtration chromatography may be used.Acceptable purity is at least 90% or at least 95% or at least 98% asassessed by analytical HPLC.

When a protein has not been mapped for CD4 epitopes or CD8 epitopes orboth, a set of SLPs that comprise the entire protein sequence may besynthesized. Each SLP will typically be about 50 aa, and consecutiveSLPs may overlap in sequence by about 25 aa. Alternatively, or inaddition, algorithms and computer programs can be used to predictsequences that will bind to MHC class I and class II molecules. Suchprograms are readily available, e.g., RANKPEP (Reche et al., HumanImmunol 63: 701, 2002), Epipredict (Jung et al., Biologicals 29: 179,2001) and MHCPred (Guan et al. Nucl Acids Res 31: 3621, 2003 and Guan etal., Appl Bioinformatics 5: 55, 2006), EpiMatrix (EpiVax, Inc.).

The sequence of an SLP may be adjusted as necessary for optimumproduction. For example, one or more amino acids at the ends of apeptide derived from a native sequence may be omitted in order toimprove solubility or stability, or to increase or decrease the overallcharge. As a specific example, a peptide sequence with a high content ofhydrophobic amino acids may be difficult to solubilize. As a guide,hydrophobic content is ideally less than 50%. Peptides containingcysteine, methionine, or tryptophan residues, especially multiple Cys,Met, or Trp residues, may be difficult to synthesize. Substitution ofanother amino acid, either a standard or a non standard amino acid, suchas hydroxyproline, gamma-aminobutyric acid, norleucine, may improvesynthesis efficiency or purity. Other considerations in designing an SLPinclude the extent of β-sheet formation, N-terminal amino acid (e.g., anN-terminal Gln can cyclize), minimizing adjacent Ser and Pro residues.

Some structural proteins that are especially useful for inclusion in apharmaceutical composition include UL19 (SEQ ID No. 4), UL19 UpperDomain Fragment (SEQ ID No. 12), UL 25 (SEQ ID No. 5) and UL47 (SEQ IDNo. 6). Structure of viral proteins may be found in MMDB (MolecularModeling Database) of NCBI. Molecular structure information is availablefor UL25 (MMDB ID: 37706, Bowman et al. J. Virol. 80:2309, 2006,incorporated in its entirety), VP5 (product of UL19) (MMDB ID: 26005,Bowman et al., EMBO J. 22: 757-765, 2003, incorporated in its entirety),VP13/14 (product of UL47) (MMDB ID: 6022), and envelope protein gD2(MMDB ID: 36244, Krummenacher et al. EMBO J. 24:4144-4153, 2005,incorporated in its entirety), ICP34.5, as well as many other HSV-2proteins. In addition, some T-cell epitopes of viral proteins are known(Koelle et al., J Virol 74:10930-10938, 2000; Muller et al., J Gen Virol90:1153-1163, 2009; Koelle et all, J Immunol 166:4049-4058, 2001;BenMohamed et al., J Virol 77:9463-9473, 2003; U.S. Pat. No. 6,855,317;P.C.T. Pub. No. WO 2004/009021, all of which references are incorporatedin their entirety).

Immunogenic fragments, variants and fusion proteins of any of these areproteins, especially UL19, UL19 Upper Domain Fragment, UL25 and UL47,are specifically contemplated for use in the immunogenic compositionsherein. Thus, the disclosure includes fragments or variants of any oneof SEQ ID NO: 4, 5, 6, or 12 that retain at least 90% amino acididentity over at least 10 contiguous amino acids thereof, or at least85% amino acid identity over at least 15 contiguous amino acids thereof.As another example, the disclosure includes immunogenic fragmentscomprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 48 or 50 contiguousamino acids of the sequence, or between about 6-10, 10-15, 15-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or more contiguousamino acids of the sequence. The disclosure also includes variantshaving at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%. 98%, or 99% identity over at least 50 contiguous amino acids of thesequence, or over at least 100 contiguous amino acids of the sequence.In some embodiments, the variant is a naturally occurring variant,preferably one that hybridizes under stringent conditions to apolynucleotide encoding any one of SEQ ID NO: 4, 5, 6 or 12.

As disclosed herein, immunogenic fragments, including peptides, of anon-envelope structural protein (e.g., UL19 peptides as set forth in SEQID Nos. 9 and 10 and UL25 peptides as set forth in SEQ ID No. 11) and ofan envelope protein (e.g., gD2 (SEQ ID Nos. 7 and 8) may be used or maybe part of a longer sequence (i.e., fragment) derived from the protein.Peptides, as used herein, refer to short sequences of amino acids,generally from at least 15 residues and generally up to about 100residues, or from about 20 residues to about 80 residues, or from about30 residues to about 70 residues. Fragments, as used herein, refer toany length of polypeptide less than full-length protein and aregenerally at least 100 amino acids long, although the size range offragments can overlap the size range of peptides (e.g., fragments fromabout 50 residues long). In particular, a UL19 Upper Domain Fragment ismissing at least 75%, 80%, 85%, 90%, 95% or all of residues 1-450 andresidues 1055-1374 of UL19. As such, the Upper Domain Fragment maybegin, for example, at any one of residues 337-451, and end at any oneof residues 1055-1294 (and is lacking at least amino acids 1-336 and1295-1374 of SEQ ID NO: 4). For example, a UL19 fragment may be fromabout residue 451 to about 1054 (SEQ ID NO:12). A UL19 Upper DomainFragment may comprise about 50, 100, 150, 200, 250, 300, 350, 400, 450,or 500 amino acids or more of SEQ ID NO: 12.

In addition, the peptides and fragments herein may be fused toheterologous peptides. Examples of heterologous peptides includesequences from other proteins (e.g., in the case of UL19, a UL19 UpperDomain Fragment may be fused to a sequence from another protein that isnot UL19), or tag sequences, such as hexa-histidine, which generallywill be located at either the N-terminus or the C-terminus. Thus, theimmunogenic fragments or variants described herein may be fused toanother peptide that enhances immunogenicity, another peptide thatserves as a tag or marker, or another peptide from another HSV-2structural protein. As such, an immunogenic polypeptide may comprise afragment consisting of a designated fragment of an HSV-2 structuralprotein. In one example, an immunogenic polypeptide comprises a fragmentof UL19 consisting of SEQ ID NO: 12 or a fragment of SEQ ID NO: 12,optionally fused to a non-UL19 peptide. In another example, animmunogenic polypeptide comprises a peptide consisting of an amino acidsequence that is at least 80% or 90% identical over 50 contiguous aminoacids of SEQ ID NO: 12, optionally fused to a non-UL19 peptide.

Surprisingly, the examples herein show that a UL19 Upper Domain Fragmenthas the ability to elicit protective antibodies to HSV-2 infection, suchthat the remainder of the UL19 protein is not needed as an immunogen.This surprising discovery is fortuitous as attempts to express fulllength UL19 have proven challenging. For example, full length UL19expression in E. coli and other expression systems, and subsequentpurification of soluble full length UL 19, has proven difficult.

Typically the proteins in a pharmaceutical composition will be otherthan a precursor protein because expression in a eukaryotic cell willtypically result in a mature protein, lacking the leader sequence (alsoknown as a signal peptide). The leader sequence of gD encompassesapproximately residues 1-25. The leader sequence of gB encompassesapproximately residues 1-22. Glycoprotein D (SEQ ID No. 2) is 393 aminoacid protein and has an extracellular region spanning approximatelyresidues 26-340, a transmembrane region spanning approximately residues341-361 and a cytoplasmic region spanning approximately residues362-393, and a number of N-linked glycosylation sites at residues 119,146, 287 (UniProtKB/Swiss-Prot accession number Q69467, version 49 ofentry and version 1 of sequence). An exemplary gD fragment (hereinalternatively referred to as gD2) comprises the sequence shown in SEQ IDNo. 3.

In some embodiments, antigenic and immunogenic fragments from envelopeglycoproteins may comprise part or all of a leader sequence, which issometimes called a signal peptide. The leader sequence is usuallyapproximately 15-20 amino acids, and in normal cellular processes, itmay be cleaved off by cellular apparatus, however, some of theglycoprotein in intact virions may have the leader sequence. Leadersequences usually have some polar amino acids at the N-terminus and theinternal amino acids are generally hydrophobic. As discussed above, theleader sequences for some of the HSV-2 envelope glycoproteins have beendetermined. For other HSV-2 envelope glycoproteins, computer programsmay be used to predict the signal peptide. Some of these programsinclude SIG-Pred (bmbpcu36.leeds.ac.uk/prot_analysis/Signal.html),PrediSi, OCTOPUS (octopus.cbr.su.se), and sigcleave(emboss.sourceforge.net/apps/cvs/emboss/apps/sigcleave.html).

A variety of techniques may be used to inhibit cleavage of the signalpeptide during cellular production of an antigenic or immunogenicfragment containing the leader sequence for use in the compositionsdescribed herein. For example, one or more of the amino acids flankingthe cleavage site may be altered to a different amino acid, resulting ina sequence that is not recognized or cleaved by cellular apparatus. Forthis method, alterations are designed based on cleavage sites known inthe art: glycine is not preferentially used in any of the positions,tyrosine, is rarely found in the first few positions after cleavagesites, whereas proline is often found in many cleavage sites except atthe +1 position and glutamine is commonly found at the +1 residue (Zhangand Henzel, Protein Sci. 13: 219, 2004). The proposed sequence may beevaluated with a prediction program to determine if cleavage is likelyto be inhibited. If cleavage is likely, then additional alterations aremade and the newly proposed sequence re-evaluated. Other techniques toinhibit cleavage of a signal peptide include addition of one or moreamino acids at the recognition and cleavage sequence, N-terminaladdition of a signal peptide and recognition sequence such that theadded signal peptide is preferentially cleaved, and production in a hostcell that lacks the machinery to cleave the signal peptide.

In certain embodiments, a fragment comprises an HSV-2 glycoprotein,including the leader sequence. In other embodiments, a fragmentcomprises a portion of a HSV-2 glycoprotein including from the leadersequence to the start of the transmembrane domain. In yet otherembodiments, a fragment comprises a portion of an HSV-2 glycoproteinincluding from the leader sequence and ending within the extracellulardomain. In other embodiments, a fragment comprises non-contiguousportions of an HSV-2 glycoprotein, in which one of the portionscomprises an antigenic epitope in the leader sequence. In yet otherembodiments, a fragment comprises non-contiguous portions of an HSV-2glycoprotein, in which the portions comprise an epitope or it comprisesportions from different HSV-2 glycoproteins, in which the portionscomprise an epitope.

Glycoprotein B (SEQ ID No. 1) has an extracellular region spanningapproximately residues 23-771, a transmembrane region spanningapproximately residues 772-792 and a cytoplasmic region spanningapproximately residues 793-904, and a number of N-linked glycosylationsites at residues 82, 136, 393, 425, 486, 671 (UniProtKB/Swiss-Protaccession number P08666, version 60 of entry and version 2 of sequence).Glycoprotein K is a 338 amino acid protein with a 30 amino acid leadersequence at its N-terminal end (Ramaswarmy and Holland, Virology186:579-587, 1992). Glycoprotein C has a predicted 27 amino acid leadersequence, glycoprotein E has a predicted 23 amino acid leader sequence,and glycoprotein L has a predicted 16 amino acid leader sequence (SignalPeptide Resource, proline.bic.nus.edu.sg, accessed 6 Oct. 2011).

Proteins or protein fragments are preferably immunogenic. An “immunogen”is capable of inducing an immune response. Immunogenic peptide sequencesare generally recognized by T cells (e.g., CD4 or CD8 T cells) in atleast some seropositive subjects. Peptide sequences can be identified byscreening peptides derived from the complete sequence, generally using aseries of overlapping peptides. A variety of assays can be used todetermine if T cells recognize and respond to a peptide. For example, achromium-release cytotoxicity assay (Kim et al., J Immunol181:6604-6615, 2008, incorporated for its assay protocol), ELISPOTassay, an intracellular cytokine staining assay and MHC multimerstaining (Novak et al. J Clin Invest 104:R63-R67, 1999; Altman et al.,Science 274:94-96, 1996) are among suitable assays. In some cases, thefragment(s) comprise immunodominant peptide sequences. Someimmunodominant epitopes have been identified for HSV-2 glycoproteins andstructural proteins (e.g., Kim et al. J Immunol 181:6604-6615, 2008;Chentoufi et al., J. Virol. 82:11792-11802, 2008; Koelle et al., ProcNatl Acad Sci USA 100: 12899-12904, 2003; all references are herebyincorporated in their entirety). Immunogenic peptides can also bepredicted by bioinformatic software (Flower, Methods in MolecularBiology vol. 409, 2007). Some exemplary programs and databases includeFRED (Feldhahn et al. Bioinformatics 15:2758-9, 2009), SVMHC (Dönnes andKohlbacher, Nucleic Acids Res 34:W1940197, 2006), AntigenDB (Ansari etal., Nucleic Acids Res 38:D847-853, 2010), TEPITOPE (Bian and HammerMethods 34:468-475, 2004),

Any of the HSV-2 proteins, including precursor proteins, mature proteinsand fragments, including peptides, can be incorporated as part of afusion protein. The fusion partner or partners can be any of the HSV-2proteins or a non-HSV-2 protein sequence. Some common reasons to usefusion proteins are to improve expression or aid in purification of theresulting protein. For example, a signal peptide sequence tailored forthe host cell of an expression system can be linked to an HSV-2 proteinor a tag sequence for use in protein purification can be linked, andsubsequently cleaved if a cleavage sequence is also incorporated.Multiple peptide epitopes from one or more of the proteins can be fusedor fragments from one or more of the proteins can be fused. For example,structural proteins or fragments of structural proteins can be linked,such as a fusion protein of VP13/14 (UL47) and major capsid protein(UL19) or UL25 and UL47 or UL25 and UL19. The segments of a fusionprotein can be in any order, that is for a fusion of UL19 and UL47,either protein can be at the N-terminus. Similarly, multiple peptideepitopes can be in any order.

Manufacture of HSV-2 proteins, including precursor proteins, fragments,and fusion proteins is generally achieved by expression in culturedcells or by chemical synthesis. (“HSV-2 proteins” is used herein toinclude all these forms.) Short fragments are commonly synthesizedchemically, either using a machine (many are commercially available) ormanually. If produced by cells, a variety of suitable expressionsystems, both prokaryotic and eukaryotic systems, are well known and maybe used. Host cells often used and suitable for production of proteinsinclude E. coli, yeast, insect, and mammalian. Expression vectors andhost cells are commercially available (e.g., Invitrogen Corp., Carlsbad,Calif., USA) or may be constructed. An exemplary vector comprises apromoter and cloning site for the sequence encoding a protein ofinterest such that the promoter and sequence are operatively linked.Other elements may be present, such as a secretion signal sequence(sometimes called a leader sequence), a tag sequence (e.g., hexa-His),transcription termination signal, an origin of replication, especiallyif the vector is replicated extra-chromosomally, and a sequence encodinga selectable product. Methods and procedures to transfect host cells arealso well known.

Expressed proteins are collected and may be used “as-is” or moretypically, analyzed and further purified. Typical procedures fordetermining purity or quantity include gel electrophoresis, Westernblotting, mass spectrometry, and ELISA. Activity of proteins isgenerally assessed in a biological assay, such as those described in theExamples. If necessary or desired, proteins may be further purified.Many purification methods are well known and include sizechromatography, anion or cation exchange chromatography, affinitychromatography, precipitation, and immune precipitation. Intended use ofthe protein will typically determine the extent of purification, withuse in humans requiring likely the highest level of purity.

B. Agents that Activate Innate Immunity

The innate immune system comprises cells that provide defense in anon-specific manner to infection by other organisms. Innate immunity isan immediate defense but it is not long-lasting or protective againstfuture challenges. Immune system cells that generally have a role ininnate immunity are phagocytic, such as macrophages and dendritic cells.The innate immune system interacts with the adaptive (also calledacquired) immune system in a variety of ways. Cells of the innate immunesystem can participate in antigen presentation to cells of the adaptiveimmune system, including expressing lymphokines that activate othercells, emitting chemotactic molecules that attract cells that may bespecific to the invader, and secreting cytokines that recruit andactivate cells of the adaptive immune system. The immunogenicpharmaceutical compositions disclosed herein include an agent thatactivates innate immunity in order to enhance the effectiveness of thecomposition.

Many types of agents can activate innate immunity. Organisms, likebacteria and viruses, can activate innate immunity, as can components oforganisms, chemicals such as 2′-5′ oligo A, bacterial endotoxins, RNAduplexes, single stranded RNA and other molecules. Many of the agentsact through a family of molecules—the Toll-like receptors (TLRs).Engaging a TLR can also lead to production of cytokines and chemokinesand activation and maturation of dendritic cells, components involved indevelopment of acquired immunity. The TLR family can respond to avariety of agents, including lipoprotein, peptidoglycan, flagellin,imidazoquinolines, CpG DNA, lipopolysaccharide and double stranded RNA(Akira et al. Biochemical Soc Transactions 31: 637-642, 2003). Thesetypes of agents are sometimes called pathogen (or microbe)-associatedmolecular patterns.

In one aspect, one or more adjuvants are included in the composition, inorder to provide an agent(s) that activates innate immunity. An adjuvantis a substance incorporated into or administered simultaneously withantigen that increases the immune response. A variety of mechanisms havebeen proposed to explain how different adjuvants work (e.g., antigendepots, activators of dendritic cells, macrophages). Without wishing tobe bound by theory, one mechanism involves activating the innate immunesystem, resulting in the production of chemokines and cytokines, whichin turn activate the adaptive (acquired) immune response. In particular,some adjuvants activate dendritic cells through TLRs. Thus, an adjuvantis one type of agent that activates the innate immune system that may beused in a vaccine to HSV-2. An adjuvant may act to enhance an acquiredimmune response in other ways too. Preferably the adjuvant is a TLR4agonist.

One adjuvant that may be used in the compositions described herein is amonoacid lipid A (MALA) type molecule. An exemplary MALA is MPL®adjuvant as described in, e.g., Ulrich J. T. and Myers, K. R.,“Monophosphoryl Lipid A as an Adjuvant” Chapter 21 in Vaccine Design,the Subunit and Adjuvant Approach, Powell, M. F. and Newman, M. J., eds.Plenum Press, NY 1995. Another exemplary MALA is described by thechemical formula (I):

wherein the moieties A¹ and A² are independently selected from the groupof hydrogen, phosphate, phosphate salts, carboxylate, carboxylate salts,sulfate, sulfate salts, sulfite, sulfite salts, aspartate, aspartatesalts, succinate, succinate salts, carboxymethylphosphate andcarboxymethylphosphate salts. Sodium and potassium are exemplarycounterions for the phosphate and carboxylate salts. At least one of A¹and A² is hydrogen. The moieties R¹, R², R³, R⁴, R⁵, and R⁶ areindependently selected from the group of hydrocarbyl having 3 to 23carbons, preferably a straight chain alkyl, represented by C₃-C₂₃. Foradded clarity it will be explained that when a moiety is “independentlyselected from” a specified group having multiple members, it should beunderstood that the member chosen for the first moiety does not in anyway impact or limit the choice of the member selected for the secondmoiety. The carbon atoms to which R¹, R³, R⁵ and R⁶ are joined areasymmetric, and thus may exist in either the R or S stereochemistry. Inone embodiment all of those carbon atoms are in the R stereochemistry,while in another embodiment all of those carbon atoms are in the Sstereochemistry.

“Hydrocarbyl” or “alkyl” refers to a chemical moiety formed entirelyfrom hydrogen and carbon, where the arrangement of the carbon atoms maybe straight chain or branched, noncyclic or cyclic, and the bondingbetween adjacent carbon atoms maybe entirely single bonds, i.e., toprovide a saturated hydrocarbyl, or there may be double or triple bondspresent between any two adjacent carbon atoms, i.e., to provide anunsaturated hydrocarbyl, and the number of carbon atoms in thehydrocarbyl group is between 3 and 24 carbon atoms. The hydrocarbyl maybe an alkyl, where representative straight chain alkyls include methyl,ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, includingundecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, etc.; while branched alkyls include isopropyl,sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representativesaturated cyclic hydrocarbyls include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like; while unsaturated cyclichydrocarbyls include cyclopentenyl and cyclohexenyl, and the like.Unsaturated hydrocarbyls contain at least one double or triple bondbetween adjacent carbon atoms (referred to as an “alkenyl” or “alkynyl”,respectively, if the hydrocarbyl is non-cyclic, and cycloalkeny andcycloalkynyl, respectively, if the hydrocarbyl is at least partiallycyclic). Representative straight chain and branched alkenyls includeethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl,2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like; while representative straightchain and branched alkynyls include acetylenyl, propynyl, 1-butynyl,2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like. Forexample, “C₆₋₁₁ alkyl” mean an alkyl as defined above, containing from6-11 carbon atoms, respectively.

The adjuvant of formula (I) may be obtained by synthetic methods knownin the art, for example, the synthetic methodology disclosed in PCTInternational Publication No. WO 2009/035528, which is incorporatedherein by reference, as well as the publications identified in WO2009/035528, where each of those publications is also incorporatedherein by reference. Certain of the adjuvants may also be obtainedcommercially. A preferred adjuvant is Product No. 699800 as identifiedin the catalog of Avanti Polar Lipids, Alabaster Ala., wherein R1, R3,R5 and R6 are undecyl and R2 and R4 are tridecyl.

In various embodiments of the invention, the adjuvant has the chemicalstructure of formula (I) but the moieties A1, A2, R1, R2, R3, R4, R5,and R6 are selected from A1 being phosphate or phosphate salt and A2 ishydrogen; and R1, R3, R5 and R6 are selected from C7-C15 alkyl; and R2and R4 are selected from C9-C17 hydrocarbyl. In a preferred embodimentof the invention, the GLA used in the examples herein has the structuralformula set forth in FIG. 1, wherein R1, R3, R5 and R6 are undecyl andR2 and R4 are tridecyl.

The MALA adjuvants described above are a preferred adjuvant class foruse in the immunogenic pharmaceutical compositions described herein.However, any of the following adjuvants may also be used alone, or incombination with an MALA adjuvant, in formulating an immunogenicpharmaceutical composition.

The adjuvant may be alum, where this term refers to aluminum salts, suchas aluminum phosphate (AlPO4) and aluminum hydroxide (Al(OH)₃). Whenalum is used as the adjuvant or as a co-adjuvant, the alum may bepresent, in a dose of immunogenic pharmaceutical composition in anamount of about 100 to 1,000 μg, or 200 to 800 μg, or 300 to 700 μg or400 to 600 μg. If the adjuvant of formula (I) is co-formulated withalum, the adjuvant of formula (I) is typically present in an amount lessthan the amount of alum, in various aspects the adjuvant of formula (I),on a weight basis, is present at 0.1-1%, or 1-5%, or 1-10%, or 1-100%relative to the weight of alum. In one aspect of the invention, thecomposition excludes the presence of alum.

The adjuvant may be an emulsion having vaccine adjuvant properties. Suchemulsions include oil-in-water emulsions. Freund's incomplete adjuvant(IFA) is one such adjuvant Another suitable oil-in-water emulsion isMF-59™ adjuvant which contains squalene, polyoxyethylene sorbitanmonooleate (also known as Tween™ 80 surfactant) and sorbitan trioleate.Squalene is a natural organic compound originally obtained from sharkliver oil, although also available from plant sources (primarilyvegetable oils), including amaranth seed, rice bran, wheat germ, andolives. Other suitable emulsion adjuvants are Montanide™ adjuvants(Seppic Inc., Fairfield N.J.) including Montanide™ ISA 50V which is amineral oil-based adjuvant, Montanide™ ISA 206, and Montanide™ IMS 1312.While mineral oil may be present in the adjuvant, in one embodiment, theoil component(s) of the compositions of the present invention are allmetabolizable oils.

The adjuvant may be AS02™ adjuvant or AS04™ adjuvant. AS02™ adjuvant isan oil-in-water emulsion that contains both MPL™ adjuvant and QS-21™adjuvant (a saponin adjuvant discussed elsewhere herein). AS04™ adjuvantcontains MPL™ adjuvant and alum. The adjuvant may be Matrix-M™ adjuvant.

The adjuvant may be a saponin such as those derived from the bark of theQuillaja saponaria tree species, or a modified saponin, see, e.g., U.S.Pat. Nos. 5,057,540; 5,273,965; 5,352,449; 5,443,829; and 5,560,398. Theproduct QS-21™ adjuvant sold by Antigenics, Inc. Lexington, Mass. is anexemplary saponin-containing co-adjuvant that may be used with theadjuvant of formula (1). Related to the saponins is the ISCOM™ family ofadjuvants, originally developed by Iscotec (Sweden) and typically formedfrom saponins derived from Quillaja saponaria or synthetic analogs,cholesterol, and phospholipid, all formed into a honeycomb-likestructure.

The adjuvant may be a cytokine that functions as an adjuvant, see, e.g.,Lin R. et al. Clin. Infec. Dis. 21(6):1439-1449 (1995); Taylor, C. E.,Infect. Immun. 63(9):3241-3244 (1995); and Egilmez, N. K., Chap. 14 inVaccine Adjuvants and Delivery Systems, John Wiley & Sons, Inc. (2007).In various embodiments, the cytokine may be, e.g.,granulocyte-macrophage colony-stimulating factor (GM-CSF); see, e.g.,Change D. Z. et al. Hematology 9(3):207-215 (2004), Dranoff, G. Immunol.Rev. 188:147-154 (2002), and U.S. Pat. No. 5,679,356; or an interferon,such as a type I interferon, e.g., interferon-α (IFN-α) or interferon-β(IFN-β), or a type II interferon, e.g., interferon-γ (IFN-γ), see, e.g.,Boehm, U. et al. Ann. Rev. Immunol. 15:749-795 (1997); andTheofilopoulos, A. N. et al. Ann. Rev. Immunol. 23:307-336 (2005); aninterleukin, specifically including interleukin-1α (IL-1α),interleukin-10 (IL-1β), interleukin-2 (IL-2); see, e.g., Nelson, B. H.,J. Immunol. 172(7):3983-3988 (2004); interleukin-4 (IL-4), interleukin-7(IL-7), interleukin-12 (IL-12); see, e.g., Portielje, J. E., et al.,Cancer Immunol. Immunother. 52(3): 133-144 (2003) and Trinchieri. G.Nat. Rev. Immunol. 3(2):133-146 (2003); interleukin-15 (1′-15),interleukin-18 (IL-18); fetal liver tyrosine kinase 3 ligand (Flt3L), ortumor necrosis factor α (TNFα).

The adjuvant may be unmethylated CpG dinucleotides, optionallyconjugated to the antigens described herein.

Examples of immunopotentiators that may be used in the practice of themethods described herein as co-adjuvants include: MPL™; MDP andderivatives; oligonucleotides; double-stranded RNA; alternativepathogen-associated molecular patterns (PAMPS); saponins; small-moleculeimmune potentiators (SMIPs); cytokines; and chemokines.

In various embodiments, the co-adjuvant is MPL™ adjuvant, which iscommercially available from GlaxoSmithKline (originally developed byRibi ImmunoChem Research, Inc. Hamilton, Mont.). See, e.g., Ulrich andMyers, Chapter 21 from Vaccine Design: The Subunit and AdjuvantApproach, Powell and Newman, eds. Plenum Press, New York (1995). Relatedto MPL™ adjuvant, and also suitable as co-adjuvants for use in thecompositions and methods described herein, are AS02™ adjuvant and AS04™adjuvant. AS02™ adjuvant is an oil-in-water emulsion that contains bothMPL™ adjuvant and QS-21™ adjuvant (a saponin adjuvant discussedelsewhere herein). AS04™ adjuvant contains MPL™ adjuvant and alum. MPL™adjuvant is prepared from lipopolysaccharide (LPS) of Salmonellaminnesota R595 by treating LPS with mild acid and base hydrolysisfollowed by purification of the modified LPS.

When two adjuvants are utilized in combination, the relative amounts ofthe two adjuvants may be selected to achieve the desired performanceproperties for the composition which contains the adjuvants, relative tothe antigen alone. For example, the adjuvant combination may be selectedto enhance the antibody response of the antigen, and/or to enhance thesubject's innate immune system response. Activating the innate immunesystem results in the production of chemokines and cytokines, which inturn may activate an adaptive (acquired) immune response. An importantconsequence of activating the adaptive immune response is the formationof memory immune cells so that when the host re-encounters the antigen,the immune response occurs quicker and generally with better quality.

The adjuvant(s) may be pre-formulated prior to their combination withthe HSV-2 proteins. In one embodiment, an adjuvant may be provided as astable aqueous suspension of less than 0.2 um and may further compriseat least one component selected from the group consisting ofphospholipids, fatty acids, surfactants, detergents, saponins,fluorodated lipids, and the like. The adjuvant(s) may be formulated inan oil-in-water emulsion in which the adjuvant is incorporated in theoil phase. For use in humans, the oil is preferably metabolizable. Theoil may be any vegetable oil, fish oil, animal oil or synthetic oil; theoil should not be toxic to the recipient and is capable of beingtransformed by metabolism. Nuts (such as peanut oil), seeds, and grainsare common sources of vegetable oils. Particularly suitablemetabolizable oils include squalene(2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane), anunsaturated oil found in many different oils, and in high quantities inshark-liver oil. Squalene is an intermediate in the biosynthesis ofcholesterol. In addition, the oil-in-water emulsions typically comprisean antioxidant, such as alpha-tocopherol (vitamin E, U.S. Pat. No.5,650,155, U.S. Pat. No. 6,623,739). Stabilizers, such as atriglyceride, ingredients that confer isotonicity, and other ingredientsmay be added. An exemplary oil-in-water emulsion using squalene is knownas “SE” and comprises squalene, glycerol, phosphatidylcholine orlecithin or other block co-polymer as a surfactant in an ammoniumphosphate buffer, pH 5.1, with alpha-toceraphol.

The method of producing oil-in-water emulsions is well known to a personskilled in the art. Commonly, the method comprises mixing the oil phasewith a surfactant, such as phosphatidylcholine, poloxamer, blockco-polymer, or a TWEEN80® solution, followed by homogenization using ahomogenizer. For instance, a method that comprises passing the mixtureone, two, or more times through a syringe needle is suitable forhomogenizing small volumes of liquid. Equally, the emulsificationprocess in a microfluidiser (M110S microfluidics machine, maximum of 50passes, for a period of 2 min at maximum pressure input of 6 bar (outputpressure of about 850 bar)) can be adapted to produce smaller or largervolumes of emulsion. This adaptation can be achieved by routineexperimentation comprising the measurement of the resultant emulsionuntil a preparation was achieved with oil droplets of the desireddiameter. Other equipment or parameters to generate an emulsion may alsobe used. Disclosures of emulsion compositions, and method of theirpreparation, may be found in, e.g., U.S. Pat. Nos. 5,650,155; 5,667,784;5,718,904; 5,961,970; 5,976,538; 6,572,861; and 6,630,161.

C. Pharmaceutical Compositions and Uses

1. Formulation

A claimed pharmaceutical composition comprises an HSV-2 glycoprotein oran immunogenic fragment thereof, an HSV-2 structural protein other thanan envelope glycoprotein or an immunogenic fragment thereof, an agentthat is an agonist for the innate immune system, and a pharmaceuticallyacceptable carrier. The composition may comprise more than oneglycoprotein (or fragment), more than one structural protein (orfragment) or more than one agent.

In some aspects, the pharmaceutical composition comprises an antigenicportion of an HSV glycoprotein, a pharmaceutically acceptable carrier,and optionally an agent that is an agonist for the innate immune system.The composition may comprise more than one glycoprotein portion and oneor more than one agent. The carrier may optionally have adjuvantproperties, e.g., some emulsion carriers have adjuvant properties.Although herein primarily the HSV glycoproteins that are discussed arefrom HSV-2, glycoproteins from HSV-1 may also be used.

In certain embodiments, the glycoprotein or the structural protein orboth may be a precursor protein, a mature protein, a fragment, a fusionprotein, or a peptide. The glycoprotein and structural protein elementsmay be part of the same or different fusion proteins. Similarly, ifthere is more than one glycoprotein or more than one structural protein,they may be part of a single fusion protein or parts of separate fusionproteins. If there is more than one glycoprotein or more than onestructural protein, each of the more than one proteins can be aprecursor protein, mature protein, fragment, etc. that is, for example,two glycoproteins may comprise a fragment and a peptide or for example,two different fragments of the same glycoprotein or for example, twofragments of different glycoproteins.

The amount of each of the proteins or immunologic fragments in eachvaccine dose typically ranges from about 0.5 μg to about 50 μg, or about0.5 μg, about 1.0 μg, about 2 μg, about 5 μg, about 10 μg, about 15 μg,about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 75 μg, about100 μg, or about 150 μg or about 200 μg or about 250 μg or any othersuitable amount that would be determined to provide efficacy againstHSV-2. The proteins or immunologic fragments may be present in a varietyof ratios, including equimolar ratios, which provides equal epitoperepresentation, and equimass ratios, which provides equal mass of eachindividual protein. Equimolar and equimass ratios that are within about20% (e.g., 0.8:1.2), or within about 10% (e.g., 0.9:1.1) or within about5% (e.g., 0.95:1.05) of equivalence are still considered to be equimolaror equimass. The dose will typically be determined by pharmacologicalactivity of the composition, purpose (therapeutic or prophylactic), andthe size and condition of the subject.

The proteins may be supplied as a solution, but can also be desiccated(dry) in which case, a user adds the necessary liquid. Typically,additives such as buffers, stabilizers, tonicity agents, surfactants,preservatives, carriers, and other non-active ingredients will also bepresent. The additives are typically pharmaceutically acceptable andbio-compatible. Preferably, the additives, immunogens, agents, etc. aresubstantially free of other endotoxins, toxic compounds, andcontaminants that can cause unwanted side-effects. Formulations may varyaccording to the route of administration. For example, a formulation foradministration by i.m. injection will generally be isotonic and aqueous,while a formulation for oral administration may be encapsulated as aslow-release form or contain flavors. Formations for aerosoladministration will generally be packaged under pressure and contain apropellant.

The agent, which may be an adjuvant, may be provided as a solution,desiccated, or emulsified, generally as a stable oil-in-water emulsion.In one embodiment, an agent, may be provided as a stable aqueoussuspension of less than 0.2 um and may further comprise at least onecomponent selected from the group consisting of phospholipids, fattyacids, surfactants, detergents, saponins, fluorodated lipids, and thelike. Such a stable aqueous formulation may be a micellar formulation.In another embodiment, the agent may be formulated in a manner which canbe aerosolized, either as a powder or liquid formulation.

Any of these may also comprise buffers, stabilizers, preservatives,carriers, or other non-active ingredients. The additives are typicallypharmaceutically acceptable and bio-compatible. More than one agent maybe present, and one, some or all of the agents may also be an adjuvantor co-adjuvant. In addition, an adjuvant, or co-adjuvant, that is notalso an agent may also be provided. Antigen depots, such as oils or atleast some oil emulsions may also be present.

The amount of an adjuvant agent such as GLA or another MALA adjuvant istypically about 0.5 μg, about 1 μg, about 2 μg, about 2.5 μg, about 5μg, about 7.5 μg, about 10 μg, about 15 μg, about 20 μg or about 25 μg.An emulsion, such as SE, may be present at about 0.1%, about 0.5%, about1.0%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%,about 7.5% or about 10%

The agent and proteins may be provided in separate containers and mixedon-site or pre-mixed. In addition, the proteins may be presented inseparate containers or combined in a single container. The agent andproteins may be provided in a concentrated form and provided with adiluent. Suitable diluents include saline and PBS. A container can avial, ampoule, tube, well of a multi-well device, reservoir, syringe orany other kind of container. The container or containers may be providedas a kit. If one or more of the containers comprises desiccatedingredients the liquids for reconstitution may be provided in the kit aswell or provided by the user. The amount of solution in each containeror that is added to each container is commensurate with the route ofadministration and how many doses are in each container. A vaccine givenby injection is typically from about 0.1 ml to about 2.0 ml, while avaccine that is given orally may be a larger volume, from about 1 ml toabout 10 ml for example. Suitable volumes may also vary according to thesize and age of the subject.

2. Administration

The composition may be used for treatment of an HSV-2 infection insubjects. As used herein, “treatment” is a clinical intervention thatmay be therapeutic or prophylactic. In therapeutic applications,pharmaceutical compositions or mendicants are administered to a subjectsuspected of having or known to have an HSV-2 infection. The compositionis given in an amount sufficient to generate (induce) an immune responsethat can cure, or at least partially arrest, the symptoms of the diseaseand its complications. In prophylactic applications, pharmaceuticalcompositions or mendicants are administered to a subject susceptible to,or otherwise at risk of, an HSV-2 infection in an amount sufficient toinduce an immune response that will inhibit infection or reduce the riskor delay the outset of the disease or ameliorate one or more of theeffects of infection. An amount adequate to accomplish this is definedas a therapeutically- or pharmaceutically-effective dose. Such an amountcan be administered as a single dosage or can be administered accordingto a regimen, whereby it is effective. The amount can cure a diseasebut, typically, is administered in order to ameliorate the symptoms of adisease, or to effect prophylaxis of a disease or disorder fromdeveloping.

In both therapeutic and prophylactic regimes, agents are usuallyadministered in several dosages until a sufficient immune response hasbeen achieved. Typically, the immune response is monitored and repeateddosages are given if the immune response starts to fade. Treatment neednot completely eliminate a disease, nor need it completely prevent asubject from becoming ill with the disease or disorder. In someembodiments, only a single dosage is administered. More often, multipledosages will be administered. Generally, the first dosage is called a“priming” dosage and the second and subsequence dosages are called“boosting” dosages. Multiple dosages may consist of two administrations,of three administrations, of four administrations, and at times, of fiveor more administrations. Ideally, the number is one or twoadministrations. When multiple administrations are provided, the timingof the second, and subsequent, administrations will generally be atleast two weeks following the last administration, and may be at leastone month, two months, three months, six months, or 1 year following thelast administration. Ideally, an immune response is monitored in orderto determine if multiple dosages would be advantageous. The multipledosages may contain equivalent amount of immunogens and agonist or maycontain different amounts of these ingredients. For example, a boostingdosage may comprise lower amounts of immunogens. Furthermore, additivesmay differ between dosages.

In some embodiments, the priming composition that is administered to thesubject is a live attenuated HSV-2 virus and the boosting compositionthat is administered to the subject is any composition claimed ordescribed herein. In some embodiments, the priming composition that isadministered to the subject is any composition claimed or describedherein and the boosting composition that is administered to the subjectis a live attenuated HSV-2 virus.

Whether used as a prophylactic or as a therapeutic, administrationpreferably raises an immune response to HSV-2. The immune response canbe humoral (antibody mediated) or cellular (typically, although notexclusively T cell mediated) or both. The immunized subject may alsohave activated monocytes, macrophages, NK cells, dendritic cells, andother innate immune cell types. Assays for an immune response aredescribed herein and are well known by one of average skill.

Vaccine is administered at a dose sufficient to effect a beneficialtherapeutic response (therapeutically effective dose) e.g., effectiveimmune response to ameliorate, alleviate, cure or partially amelioratesymptoms of disease or infection, or prophylactic response, e.g.,prevent infection or disease symptoms. Indicators of a beneficialtherapeutic response is fewer herpes lesions in any given outbreak or alower number of lesions on average, or less frequent outbreaks. Otherindicators include smaller lesions, lesions that heal more quickly,inure less pain. Still other indicators are development of antibodies toHSV-2 vaccine components, in particular presence of antibodies to HSV-2envelope glycoproteins, e.g., antibodies to gD2, and also particularlydevelopment of neutralizing antibodies. There are many well knownprocedures to detect and quantify antibodies, including ELISA andinhibition of virus infection (neutralization) assays. In oneimplementation, the ELISA assay is performed by coating wells of amulti-well plate with gD2 protein, capturing gD2-specific antibody fromserum onto the plates, detecting the gD2-specific antibody with labeledanti-human antibody, followed by a readout of the label. Label can beradioactive, but is more usually an enzyme, such as horse radishperoxidase, that converts a substrate to one that can be detectedcolorimetrically. An exemplary HSV neutralization assay is based on aplaque assay in which neutralizing antibody is detected by inhibition ofplaque formation. Other indicators include an increased amount orfunction or frequency of CD8 or CD4 T cells responsive to HSV-2, areduction in virus shedding, reduction in viral transmission to sexualpartners, and reduction of size or frequency or both of symptomaticlesions.

Assays for T cell function include IFN-γ ELISPOT and ICS (intracellularcytokine staining). The ELISPOT assay detecting interferon-gamma iswidely used to quantize CD4 and CD8 T cell responses to candidatevaccines. The ELISPOT assay is based on the principle of the ELISAdetecting antigen-induced secretion of cytokines trapped by animmobilized antibody and visualized by an enzyme-coupled secondantibody. ICS is a routinely used method to quantify cytotoxic T cellsby virtue of cytokine expression following stimulation with agonists,such as antibodies to T cell surface molecules or peptides that bind MHCClass molecules. Exemplary procedures of ICS and ELISPOT are describedbelow.

Subjects to receive the vaccine include both HSV-2 seropositive andHSV-2 seronegative individuals. For seropositive individuals, thevaccine is intended to be therapeutic. For seronegative individuals, thevaccine is intended to be protective. In some cases, subjects areseropositive for HSV-1 and in other cases, are seronegative for HSV-1,independent of HSV-2 status. That is, subjects may include those who areHSV-1 seropositive/HSV-2 seropositive, HSV-1 seronegative/HSV-2seropositive, HSV-1 seropositive/HSV-2 seronegative, HSV-1seronegative/HSV-2 seronegative. Moreover, subjects include human andother mammalian subjects that can be infected by HSV-2.

The vaccine can be administered by any suitable delivery route, such asintradermal, mucosal (e.g., intranasal, oral), intramuscular,subcutaneous, sublingual, rectal, and vaginal. Other delivery routes arewell known in the art.

The intramuscular route is one suitable route for the composition.Suitable i.m. delivery devices include a needle and syringe, aneedle-free injection device (for example Biojector, Bioject, Oreg.USA), or a pen-injector device, such as those used in self-injections athome to deliver insulin or epinephrine. Intradermal and subcutaneousdelivery are other suitable routes. Suitable devices include a syringeand needle, syringe with a short needle, and jet injection devices.

The composition may be administered by a mucosal route, e.g.,intranasally. Many intranasal delivery devices are available and wellknown in the art. Spray devices are one such device. Oral administrationcan as simple as providing a solution for the subject to swallow.

Vaccine may be administered at a single site or at multiple sites. If atmultiple sites, the route of administration may be the same at eachsite, e.g., injection in different muscles, or may be different, e.g.,injection in a muscle and intranasal spray. Furthermore, the vaccine maybe administered at a single time point or multiple time points.Generally if administered at multiple time points, the time betweendoses has been determined to improve the immune response.

Recombinant Expression Vectors, Viral Vectors, and Virus-Like Particles

In one embodiment, recombinant expression vectors are provided thatcomprise a polynucleotide sequence encoding at least one HSV2 immunogenthat induces an immune response to the immunogen and to its respectivedesignated antigen. To obtain efficient transcription and translation ofthe immunogen, the encoding polynucleotide sequences in each vectorinclude at least one appropriate expression control sequence (alsocalled a regulatory expression sequence or feature) (e.g., promoter,enhancer, leader), which are described in greater detail herein, that isoperatively linked to the encoding polynucleotide sequence(s). Theserecombinant expression vectors are thus provided for directingexpression of the immunogen or for directing co-expression of at leasttwo immunogens in any appropriate host cell that has been transformed,transduced, or transfected with the recombinant expression vector orvector particle containing the recombinant expression vector.

The recombinant expression vectors described herein may encode one ormore HSV-2 immunogens (i.e., at least one, at least two, at least threeimmunogens, etc.), which immunogens are described in greater detailherein. In particular embodiments, at least one, two, or three, or moreimmunogens from HSV-2 may be encoded by a recombinant expression vector.By way of example, an immunogen may be an HSV-2 protein, such as UL19(e.g., UL19 Upper Domain Fragment or an immunogenic fragment or variantthereof) and/or gD, (or an immunogenic fragment or variant thereof)and/or UL47 (or an immunogenic fragment or variant thereof), or may beanother immunogenic fragment or region of the HSV-2 protein.

A. Recombinant Production of Protein

A recombinant expression vector that comprises a polynucleotide sequencethat encodes an immunogen may be used for production of the immunogen.Recombinant expression vectors include at least one regulatoryexpression sequence, such as a promoter or enhancer, that is operativelylinked to the polynucleotide encoding the immunogen. Each of theexpression vectors may be used to transform, transducer, or transfect anappropriate host cell for recombinant production of a respectiveimmunogen. Suitable host cells for production of the immunogen includeprokaryotes, yeast and higher eukaryotic cells (e.g., CHO and COS). Theimmunogen may each be isolated from the respective host cell or hostcell culture using any one of a variety of isolation methods (e.g.,filtration, diafiltration, chromatography (including affinitychromatography, high pressure liquid chromatography), and preparativeelectrophoresis) known and routinely practiced in the protein art. Incertain embodiments, as described herein, the isolated immunogen maythen be formulated with a pharmaceutically suitable excipient to providean immunogenic composition.

Particular methods for producing polypeptides recombinantly aregenerally well known and routinely used. For example, molecular biologyprocedures are described by Sambrook et al. (Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York,1989; see also Sambrook et al., 3rd ed., Cold Spring Harbor Laboratory,New York, (2001)). DNA sequencing can be performed as described inSanger et al. (Proc. Natl. Acad. Sci. USA 74:5463 (1977)) and theAmersham International plc sequencing handbook and includingimprovements thereto.

B. Recombinant Expression Vectors for Delivery of Protein to Subjects

Recombinant expression vectors may be used for expression of any one ormore of the immunogens described herein. In particular embodiments, therecombinant expression vector is delivered to an appropriate cell (forexample, an antigen-presenting cell i.e., a cell that displays apeptide/MHC complex on its cell surface, such as a dendritic cell) ortissue (e.g., lymphoid tissue) that will induce the desired immuneresponse (i.e., a specific humoral response (i.e., B cell response)and/or induction of a specific cell-medicated immune response, which mayinclude an immunogen-specific CD4 and/or CD8 T cell response, which CD8T cell response may include a cytotoxic T cell (CTL) response). Therecombinant expression vectors may therefore also include, for example,lymphoid tissue-specific transcriptional regulatory elements (TRE) suchas a B lymphocyte, T lymphocyte, or dendritic cell specific TRE.Lymphoid tissue specific TRE are known in the art (see, e.g., Thompsonet al., Mol. Cell. Biol. 12, 1043-53 (1992); Todd et al., J. Exp. Med.177, 1663-74 (1993); Penix et al., J. Exp. Med. 178:1483-96 (1993)).

In a particular embodiment, the recombinant expression vector is plasmidDNA or cosmid DNA. Plasmid DNA or cosmid DNA containing one or morepolynucleotides encoding an immunogen as described herein is readilyconstructed using standard techniques well known in the art. The vectorgenome may be typically constructed in a plasmid form that can then betransfected into a packaging or producer cell line. The plasmidgenerally comprises sequences useful for replication of the plasmid inbacteria. Such plasmids are well known in the art. In addition, vectorsthat include a prokaryotic origin of replication may also include a genewhose expression confers a detectable or selectable marker such as adrug resistance. Typical bacterial drug resistance products are thosethat confer resistance to ampicillin or tetracycline. For analysis toconfirm that the correct nucleotide sequences are incorporated inplasmids, the plasmid may be replicated in E. coli, purified, andanalyzed by restriction endonuclease digestion and/or its nucleotidesequence determined by conventional methods.

C. Viral Vectors

In other particular embodiments, the recombinant expression vector is aviral vector. Exemplary recombinant expression viral vectors include alentiviral vector genome, poxvirus vector genome, vaccinia virus vectorgenome, adenovirus vector genome, adenovirus-associated virus vectorgenome, herpes virus vector genome, and alpha virus vector genome. Viralvectors may be live, attenuated, replication conditional or replicationdeficient, and typically is a non-pathogenic (defective), replicationcompetent viral vector.

By way of example, in a specific embodiment, when the viral vector is avaccinia virus vector genome, the polynucleotide encoding an immunogenof interest may be inserted into a non-essential site of a vacciniaviral vector. Such non-essential sites are described, for example, inPerkus et al., Virology 152:285 (1986); Hruby et al., Proc. Natl. Acad.Sci. USA 80:3411 (1983); Weir et al., J. Virol. 46:530 (1983). Suitablepromoters for use with vaccinia viruses include but are not limited toP7.5 (see, e.g., Cochran et al., J. Virol. 54:30 (1985); P11 (see, e.g.,Bertholet, et al., Proc. Natl. Acad. Sci. USA 82:2096 (1985)); and CAE-1(see, e.g., Patel et al., Proc. Natl. Acad. Sci. USA 85:9431 (1988)).Highly attenuated strains of vaccinia are more acceptable for use inhumans and include Lister, NYVAC, which contains specific genomedeletions (see, e.g., Guerra et al., J. Virol. 80:985-98 (2006);Tartaglia et al., AIDS Research and Human Retroviruses 8:1445-47(1992)), or MVA (see, e.g., Gheradi et al., J. Gen. Virol. 86:2925-36(2005); Mayr et al., Infection 3:6-14 (1975)). See also Hu et al. (J.Virol. 75:10300-308 (2001), describing use of a Yaba-Like disease virusas a vector for cancer therapy); U.S. Pat. Nos. 5,698,530 and 6,998,252.See also, e.g., U.S. Pat. No. 5,443,964. See also U.S. Pat. Nos.7,247,615 and 7,368,116.

In certain embodiments, an adenovirus vector or adenovirus-associatedvirus vector may be used for expressing an immunogen of interest.Several adenovirus vector systems and methods for administering thevectors have been described (see, e.g., Molin et al., J. Virol.72:8358-61 (1998); Narumi et al., Am J. Respir. Cell Mol. Biol.19:936-41 (1998); Mercier et al., Proc. Natl. Acad. Sci. USA 101:6188-93(2004); U.S. Pat. Nos. 6,143,290; 6,596,535; 6,855,317; 6,936,257;7,125,717; 7,378,087; 7,550,296).

Retroviral vector genomes may include those based upon murine leukemiavirus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses,simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV),and combinations (see, e.g., Buchscher et al., J. Virol. 66:2731-39(1992); Johann et al., J. Virol. 66:1635-40 (1992); Sommerfelt et al.,Virology 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-78 (1989);Miller et al., J. Virol. 65:2220-24 (1991); Miller et al., Mol. Cell.Biol. 10:4239 (1990); Kolberg, NIH Res. 4:43 1992; Cornetta et al., Hum.Gene Ther. 2:215 (1991)).

D. Lentiviral Vectors

In a more specific embodiment, the recombinant expression viral vectoris a lentiviral vector genome. The genome can be derived from any of alarge number of suitable, available lentiviral genome based vectors,including those identified for human gene therapy applications (see,e.g., Pfeifer et al., Annu. Rev. Genomics Hum. Genet. 2:177-211 (2001)).Suitable lentiviral vector genomes include those based on HumanImmunodeficiency Virus (HIV-1), HIV-2, feline immunodeficiency virus(FIV), equine infectious anemia virus, Simian Immunodeficiency Virus(SIV), and maedi/visna virus. A desirable characteristic of lentivirusesis that they are able to infect both dividing and non-dividing cells,although target cells need not be dividing cells or be stimulated todivide. Generally, the genome and envelope glycoproteins will be basedon different viruses, such that the resulting viral vector particle ispseudotyped. Safety features of the vector genome are desirablyincorporated. Safety features include self-inactivating LTR and anon-integrating genome. Exemplary vectors contain a packaging signal(psi), a Rev-responsive element (RRE), splice donor, splice acceptor,central polypurine tract (cPPT), and WPRE element. In certain exemplaryembodiments, the viral vector genome comprises sequences from alentivirus genome, such as the HIV-1 genome or the SIV genome. The viralgenome construct may comprise sequences from the 5′ and 3′ LTRs of alentivirus, and in particular may comprise the R and U5 sequences fromthe 5′ LTR of a lentivirus and an inactivated or self-inactivating 3′LTR from a lentivirus. The LTR sequences may be LTR sequences from anylentivirus from any species. For example, they may be LTR sequences fromHIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTRsequences.

The vector genome may comprise an inactivated or self-inactivating 3′LTR (see, e.g., Zufferey et al., J. Virol. 72: 9873, 1998; Miyoshi etal., J. Virol. 72:8150, 1998; both of which are incorporated in theirentirety). A self-inactivating vector generally has a deletion of theenhancer and promoter sequences from the 3′ long terminal repeat (LTR),which is copied over into the 5′ LTR during vector integration. In oneinstance, the U3 element of the 3′ LTR contains a deletion of itsenhancer sequence, the TATA box, Sp1 and NF-kappa B sites. As a resultof the self-inactivating 3′ LTR, the provirus that is generatedfollowing entry and reverse transcription will comprise an inactivated5′ LTR. The rationale is to improve safety by reducing the risk ofmobilization of the vector genome and the influence of the LTR on nearbycellular promoters. The self-inactivating 3′ LTR may be constructed byany method known in the art.

Optionally, the U3 sequence from the lentiviral 5′ LTR may be replacedwith a promoter sequence in the viral construct, such as a heterologouspromoter sequence. This can increase the titer of virus recovered fromthe packaging cell line. An enhancer sequence may also be included. Anyenhancer/promoter combination that increases expression of the viral RNAgenome in the packaging cell line may be used. In one example, the CMVenhancer/promoter sequence is used (see, e.g., U.S. Pat. Nos. 5,385,839and 5,168,062).

In certain embodiments, the risk of insertional mutagenesis is minimizedby constructing the lentiviral vector genome to be integrationdefective. A variety of approaches can be pursued to produce anon-integrating vector genome. These approaches entail engineering amutation(s) into the integrase enzyme component of the pol gene, suchthat it encodes a protein with an inactive integrase. The vector genomeitself can be modified to prevent integration by, for example, mutatingor deleting one or both attachment sites, or making the 3′ LTR-proximalpolypurine tract (PPT) non-functional through deletion or modification.In addition, non-genetic approaches are available; these includepharmacological agents that inhibit one or more functions of integrase.The approaches are not mutually exclusive, that is, more than one ofthem can be used at a time. For example, both the integrase andattachment sites can be non-functional, or the integrase and PPT sitecan be non-functional, or the attachment sites and PPT site can benon-functional, or all of them can be non-functional.

Integrase is involved in cleavage of viral double-stranded blunt-endedDNA and joining the ends to 5′-phosphates in the two strands of achromosomal target site. Integrase has three functional domains:N-terminal domain, which contains a zinc-binding motif (HHCC); thecentral domain core, which contains the catalytic core and a conservedDD35E motif (D64, D116, E152 in HIV-1); and a C-terminal domain, whichhas DNA binding properties. Point mutations introduced into integraseare sufficient to disrupt normal function. Many integrase mutations havebeen constructed and characterized (see, e.g., Philpott and Thrasher,Human Gene Therapy 18:483, 2007; Apolonia, Thesis submitted toUniversity College London, April 2009, pp, 82-97; Engelman et al., J.Virol. 69: 2729, 1995; Nightingale et al., Mol. Therapy, 13: 1121,2006). The sequence encoding the integrase protein can be deleted ormutated to render the protein inactive, preferably without significantlyimpairing reverse transcriptase activity or nuclear targeting, therebyonly preventing integration of the provirus into the target cell genome.Acceptable mutations can reduce integrase catalysis, strand transfer,binding to att sites, binding to host chromosomal DNA, and otherfunctions. For example, a single aspartic acid to asparaginesubstitution at residue 35 of HIV or SW integrase completely abolishesviral DNA integration. Deletions of integrase will generally be confinedto the C-terminal domain. Deletion of coding sequence for residues235-288 result in a useful non-functional integrase (see, e.g., Engelmanet al., J. Virol. 69:2729, 1995). As further examples, mutations can begenerated, for example, Asp64 (residue numbers are given for HIV-1,corresponding residue numbers for integrase from other lentiviruses orretroviruses can be readily determined by one of ordinary skill) (e.g.,D64E, D64V), Asp116 (e.g., D116N), Asn120 (e.g., N120K), Glu152, Gln148(e.g., Q148A), Lys156, Lys159, Trp235 (e.g., W235E), Lys264 (e.g.,K264R), Lys266 (e.g., K266R), Lys273 (e.g., K273R). Other mutations canbe constructed and tested for integration, transgene expression, and anyother desirable parameter. Assays for these functions are well known.Mutations can be generated by any of a variety of techniques, includingsite-directed mutagenesis and chemical synthesis of nucleic acidsequence. One mutation may be made or more than one of these mutationscan be present in integrase. For example, an integrase may havemutations at two amino acids, three amino acids, four amino acids, andso on.

Alternatively or in combination with the use of integrase mutant(s), theattachment sites (att) in U3 and U5 can also be mutated. Integrase bindsto these sites and the 3′-terminal dinucleotide is cleaved at both endsof the vector genome. A CA dinucleotide is located at the recessed 3′end; the CA is required for processing, mutation of the nucleotidesblocks integration into the host chromosome. The A of the CAdinucleotide is the most critical nucleotide for integration, andmutations at both ends of the genome will give the best results (see,e.g., Brown et al., J. Virol. 73:9011 (1999)). In one exemplification,the CA at each end is changed to TG. In other exemplifications, the CAat each end is changed to TG at one end and GT at the other end. Inother exemplifications, the CA at each end is deleted; in otherexemplifications, the A of the CA is deleted at each end.

Integration can also be inhibited by mutation or deletion of polypurinetract (PPT) (see, e.g., WO 2009/076524), located proximally to the 3′LTR. The PPT is a polypurine sequence of about 15 nucleotides that canserve as a primer binding site for plus-strand DNA synthesis. In thisinstance, mutations or deletions of PPT targets the reversetranscription process. Without wishing to be held to a particularmechanism, by mutating or deleting PPT, production of linear DNA isradically reduced, and essentially only 1-LTR DNA circles are produced.Integration requires a linear double-stranded DNA vector genome, andintegration is essentially eliminated without it. As stated herein, aPPT can be made non-functional by mutation or by deletion. Typically,the entire about 15 nt PPT is deleted, although in some embodiments,shorter deletions of 14 nt, 13, nt, 12 nt, 11 nt, 10 nt, 9 nt, 8 nt, 7nt, 6 nt, 5 nt, 4 nt, 3 nt and 2 nt may be made. When mutations aremade, typically multiple mutations are made, especially in the 5′ halfof the PPT (see, e.g., McWilliams et al., J. Virol. 77:11150, 2003),although single and double mutations in the first four bases stillreduce transcription. Mutations made at the 3′ end of PPT generally havea more dramatic effect (see, e.g., Powell et al., J. Virol. 70:5288,1996).

The U3 region may comprise a PPT (polypurine tract) sequence immediatelyupstream. In certain specific embodiments, any one of at least threedifferent U3 regions (at the 3′ end) may be included in the lentiviralvector (see SEQ ID NOS:13-15). The constructs contain deletions in theU3 regions. The SIN construct has a deletion of about 130 nucleotides inthe U3 (see, e.g., Miyoshi, et al. J. Virol. 72: 8150, 1998; Yu et al.,Proc. Natl. Acad. Sci. USA 83: 3194, 1986), which removes the TATA box,thereby abolishing LTR promoter activity. The deletions in constructs703 and 704 increase expression from lentivirus vectors (see, e.g.,Bayer et al., Mol. Therapy. 16: 1968, 2008). In addition, construct 704contains a deletion of the 3′ PPT, which decreases integration of thevector (see, e.g., WO 2009/076524). See also U.S. patent applicationSer. No. 12/842,609 and International Patent Application Publication No.WO 2011/011584 (International Patent Application No. PCT/US10/042,870),which are each incorporated by reference in their entirety.

These different approaches to make a vector genome non-integrating canbe used individually or in combination. Using more than one approach maybe used to build a fail-safe vector through redundant mechanisms. Thus,PPT mutations or deletions can be combined with att site mutations ordeletions or with Integrase mutations or PPT mutations or deletions canbe combined with both att site mutations or deletions and Integrasemutations. Similarly, att site mutations or deletions and Integrasemutations may be combined with each other or with PPT mutations ordeletions.

As described herein, lentiviral vector constructs may also contain apromoter for expression in mammalian cells. Promoters, which arediscussed in greater detail herein, include, for example, the humanubiquitin C promoter (UbiC), the cytomegalovirus immediate earlypromoter (CMV), and the Rous sarcoma virus (RSV) promoter.

E. Virus-Like Particles

In various embodiments, virus-like particles (VLP) are provided thatcomprise a at least one HSV2 immunogen that induces an immune responseto the immunogen and to its respective designated antigen.

An HSV-1 or HSV-2 virus-like particle can be prepared by allowing VP5,VP19, VP23, VP22a, and the maturational protease (UL26 gene product) toself-assemble in vitro. See, for example, Newcomb et al., J. Virol,September 1994, 6059-6063.; Newcomb et al., J. Mol. Biol., 263; 432-446(1996); Thomsen et al., J Virol, April 1994, 2442-2457; all incorporatedby reference in their entirety. The virus-like particles describedherein may comprise one or more HSV-2 immunogens (i.e., at least one, atleast two, at least three immunogens, etc.), which immunogens aredescribed in greater detail herein. In particular embodiments, at leastone, two, or three, or more immunogens from HSV-2 may be enclosed in orassociated with a virus-like particle. By way of example, an immunogenmay be an HSV-2 protein, such as UL19 (e.g., UL19 Upper Domain Fragmentor an immunogenic fragment or variant thereof) and/or gD, (or animmunogenic fragment or variant thereof) and/or UL47 (or an immunogenicfragment or variant thereof), or may be another immunogenic fragment orregion of the HSV-2 protein.

Regulatory Expression Sequences

As described herein, the recombinant expression vector comprises atleast one regulatory expression sequence. In certain embodiments, whenthe recombinant expression vector comprises a viral vector genome,expression of the at least one immunogen is desired in particular targetcells. Typically, for example, in a lentiviral vector the polynucleotidesequence encoding the immunogen is located between the 5′ LTR and 3′ LTRsequences. Further, the encoding nucleotide sequence(s) is preferablyoperatively linked in a functional relationship with other genetic orregulatory sequences or features, for example transcription regulatorysequences including promoters or enhancers, that regulate expression ofthe immunogen in a particular manner. In certain instances, the usefultranscriptional regulatory sequences are those that are highly regulatedwith respect to activity, both temporally and spatially. Expressioncontrol elements that may be used for regulating the expression of theencoded polypeptides are known in the art and include, but are notlimited to, inducible promoters, constitutive promoters, secretionsignals, enhancers, and other regulatory sequences.

The polynucleotide encoding the immunogen and any other expressiblesequence is typically in a functional relationship with internalpromoter/enhancer regulatory sequences. With respect to lentiviralvector constructs, an “internal” promoter/enhancer is one that islocated between the 5′ LTR and the 3′ LTR sequences in the viral vectorand is operatively linked to the encoding polynucleotide sequence ofinterest. The internal promoter/enhancer may be any promoter, enhanceror promoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship. A “functionalrelationship” and “operatively linked” mean, without limitation, thatthe sequence is in the correct location and orientation with respect tothe promoter and/or enhancer such that the sequence of interest will beexpressed when the promoter and/or enhancer is contacted with theappropriate molecules.

The choice of an internal promoter/enhancer is based on the desiredexpression pattern of the immunogen and the specific properties of knownpromoters/enhancers. Thus, the internal promoter may be constitutivelyactive. Non-limiting examples of constitutive promoters that may be usedinclude the promoter for ubiquitin (see, e.g., U.S. Pat. No. 5,510,474;WO 98/32869); CMV (see, e.g., Thomsen et al., Proc. Natl. Acad. Sci. USA81:659, 1984; U.S. Pat. No. 5,168,062); beta-actin (Gunning et al. 1989Proc. Natl. Acad. Sci. USA 84:4831-4835); and pgk (see, for example,Adra et al. 1987 Gene 60:65-74; Singer-Sam et al. 1984 Gene 32:409-417;and Dobson et al. 1982 Nucleic Acids Res. 10:2635-2637).

Alternatively, the promoter may be a tissue specific promoter. In someembodiments, the promoter is a target cell-specific promoter. Targetingdendritic cells may enhance the immune response, particularly thecellular cytotoxic response that is useful for immunity for HSV-2. Forexample, the promoter can be from any product expressed by dendriticcells, including CD11c, CD103, TLRs, DC-SIGN, BDCA-3, DEC-205, DCIR2,mannose receptor, Dectin-1, Clec9A, MHC class II. In addition, promotersmay be selected to allow for inducible expression of the immunogen. Anumber of systems for inducible expression are known in the art,including the tetracycline responsive system, the lac operator-repressorsystem, as well as promoters responsive to a variety of environmental orphysiological changes, including heat shock, metal ions, such asmetallothionein promoter, interferons, hypoxia, steroids, such asprogesterone or glucocorticoid receptor promoter, radiation, such asVEGF promoter. A combination of promoters may also be used to obtain thedesired expression of each of the immunogen-encoding polynucleotidesequences. The artisan of ordinary skill will be able to select apromoter based on the desired expression pattern of the polynucleotidesequence in the organism or the target cell of interest.

A recombinant expression vector, including a viral vector genome, maycomprise at least one RNA Polymerase II or III responsive promoter. Thispromoter can be operatively linked to the polynucleotide sequence ofinterest and can also be linked to a termination sequence. In addition,more than one RNA Polymerase II or III promoter may be incorporated. RNApolymerase II and III promoters are well known to persons of skill inthe art. A suitable range of RNA polymerase III promoters can be found,for example, in Paule and White, Nucleic Acids Res., Vol. 28, pp1283-1298 (2000). RNA polymerase II or III promoters also include anysynthetic or engineered DNA fragment that can direct RNA polymerase IIor III to transcribe downstream RNA coding sequences. Further, the RNApolymerase II or III (Pol II or III) promoter or promoters used as partof the viral vector genome can be inducible. Any suitable inducible PolII or III promoter can be used with the methods described herein.Particularly suited Pol II or III promoters include the tetracyclineresponsive promoters provided in Ohkawa and Taira, Human Gene Therapy,11:577-585 (2000) and in Meissner et al., Nucleic Acids Res.,29:1672-1682 (2001).

An internal enhancer may also be present in the recombinant expressionvector, including a viral vector genome, to increase expression of thepolynucleotide sequence of interest. For example, the CMV enhancer (see,e.g., Boshart et al., Cell 41:521, 1985) may be used. Many enhancers inviral genomes, such as HIV, CMV, and in mammalian genomes have beenidentified and characterized (see, e.g., publically available databasessuch as GenBank). An enhancer can be used in combination with aheterologous promoter. One of ordinary skill in the art will be able toselect the appropriate enhancer based on the desired expression pattern.

When targeting delivery of a recombinant expression vector, including aviral vector genome, to a particular target cell, the vector genome willusually contain a promoter that is recognized by the target cell andthat is operatively linked to the sequence of interest, viral components(when the vector is a viral vector), and other sequences discussedherein. A promoter is an expression control element formed by a nucleicacid sequence that permits binding of RNA polymerase and transcriptionto occur. Promoters may be inducible, constitutive, temporally active ortissue specific. The activity of inducible promoters is induced by thepresence or absence of biotic or abiotic factors. Inducible promoterscan be a useful tool in genetic engineering because the expression ofgenes to which they are operatively linked can be turned on or off atcertain stages of development of an organism, its manufacture, or in aparticular tissue. Inducible promoters can be grouped aschemically-regulated promoters, and physically-regulated promoters.Typical chemically-regulated promoters include, not are not limited to,alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) genepromoter), tetracycline-regulated promoters (e.g.,tetracycline-responsive promoter), steroid-regulated promoter (e.g., ratglucocorticoid receptor (GR)-based promoter, human estrogen receptor(ER)-based promoter, moth ecdysone receptor-based promoter, and thepromoters based on the steroid/retinoid/thyroid receptor superfamily),metal-regulated promoters (e.g., metallothionein gene-based promoters),and pathogenesis-related promoters (e.g., Arabidopsis and maizepathogen-related (PR) protein-based promoters). Typicalphysically-regulated promoters include, but are not limited to,temperature-regulated promoters (e.g., heat shock promoters), andlight-regulated promoters (e.g., soybean SSU promoter). Other exemplarypromoters are described elsewhere, for example, in patents and publishedpatent applications that can be identified by searching the U.S. Patentand Trademark Office databases.

One of skill in the art will be able to select an appropriate promoterbased on the specific circumstances. Many different promoters are wellknown in the art, as are methods for operatively linking the promoter tothe polynucleotide sequence to be expressed. Both native promotersequences and many heterologous promoters may be used to directexpression in the packaging cell and target cell. Heterologous promotersare typically used because they generally permit greater transcriptionand higher yields of the desired protein as compared to the nativepromoter.

The promoter may be obtained, for example, from the genomes of virusessuch as polyoma virus, fowlpox virus, adenovirus, bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus, and Simian Virus 40 (SV40). The promoter may also be, forexample, a heterologous mammalian promoter, for example, the actinpromoter or an immunoglobulin promoter, a heat-shock promoter, or thepromoter normally associated with the native sequence, provided suchpromoters are compatible with the target cell. In one embodiment, thepromoter is the naturally occurring viral promoter in a viral expressionsystem. In some embodiments, the promoter is a dendritic cell-specificpromoter. The dendritic cell-specific promoter can be, for example,CD11c promoter.

Transcription may be increased by inserting an enhancer sequence intothe vector(s). Enhancers are typically cis-acting elements of DNA,usually about 10 to 300 base pairs in length, that act on a promoter toincrease its transcription. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, alpha-fetoprotein, andinsulin) and from eukaryotic cell viruses. Examples include the SV40enhancer on the late side of the replication origin (base pair 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantigen-specific polynucleotide sequence, but is preferably located at asite 5′ from the promoter.

Expression vectors may also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Thesesequences are often found in the 5′ and, occasionally 3′, untranslatedregions of eukaryotic or viral DNAs or cDNAs and are well known in theart.

A recombinant expression construction, including a viral vector genome,may also contain additional genetic elements. The types of elements thatmay be included in the construct are not limited in any way and may bechosen to achieve a particular result. For example, a signal thatfacilitates nuclear entry of the recombinant expression vector or viralgenome in the target cell may be included. An example of such a signalis the HIV-1 flap signal. Additional regulatory sequences may beincluded that facilitate the characterization of the provirusintegration site in the target cell. For example, a tRNA ambersuppressor sequence may be included in the construct. An insulatorsequence, for example from chicken β-globin, may also be included in theviral genome construct. This element reduces the chance of silencing anintegrated provirus in the target cell due to methylation andheterochromatinization effects. In addition, the insulator may shieldthe internal enhancer, promoter and exogenous polynucleotide sequencesfrom positive or negative positional effects from surrounding DNA at theintegration site on the chromosome. In addition, the recombinantconstruct, including the vector genome, may contain one or more geneticelements designed to enhance expression of the gene of interest. Forexample, a woodchuck hepatitis virus responsive element (WRE) may beplaced into the construct (see, e.g., Zufferey et al. 1999. J. Virol.74:3668-81; Deglon et al., 2000. Hum. Gene Ther. 11:179-90).

When the recombinant expression vector is a viral vector genome, theviral vector genome is typically constructed in a plasmid form that maybe transfected into a packaging or producer cell line for production ofthe viral vector genome construct. The plasmid generally comprisessequences useful for replication of the plasmid in bacteria. Suchplasmids are well known in the art. In addition, vectors that include aprokaryotic origin of replication may also include a gene whoseexpression confers a detectable or selectable marker such as a drugresistance. Typical bacterial drug resistance products are those thatconfer resistance to ampicillin or tetracycline.

In certain configurations, recombinant expression vectors containpolynucleotide sequences that encode dendritic cell (DC)maturation/stimulatory factors. Exemplary stimulatory molecules includeGM-CSF, IL-2, IL-4, IL-6, IL-7, IL-15, IL-21, IL-23, TNFα, B7.1, B7.2,4-1BB, CD40 ligand (CD40L), drug-inducible CD40 (iCD40), and the like.These polynucleotides are typically under the control of one or moreregulatory elements that direct the expression of the coding sequencesin dendritic cells. In certain other particular embodiments, arecombinant expression vector is excluded that directs expression of andincludes a nucleotide sequence that encodes both an immunogen andGM-CSF. Maturation of dendritic cells contributes to successfulvaccination (see, e.g., Banchereau et al., Nat. Rev. Immunol. 5:296-306(2005); Schuler et al., Curr. Opin. Immunol. 15:138-147 (2003); Figdoret al., Nat. Med. 10:475-480 (2004)). Maturation can transform DCs fromcells actively involved in antigen capture into cells specialized for Tcell priming. For example, engagement of CD40 by CD40L on CD4-helper Tcells is a critical signal for DC maturation, resulting in potentactivation of CD8+ T cells. Such stimulatory molecules are also referredto as maturation factors or maturation stimulatory factors. Immunecheckpoints represent significant barriers to activation of functionalcellular immunity in cancer, and antagonistic antibodies specific forinhibitory ligands on T cells including CTLA4 and programmed death-1(PD-1) are examples of targeted agents being evaluated in the clinics. Asignificant tolerance mechanism in chronic infections and cancer is thefunctional exhaustion of antigen-specific T cells that express highlevels of PD-1. As the potency of therapeutic immunization has beenshown to be significantly enhanced by combination with immune checkpointcontrol, as a non-limiting example, it can be appreciated by those ofordinary skill in the art that an alternative approach to inhibitingimmune checkpoint is to inhibit the expression of programmed death (PD)ligands one and two (PD-L1/L2). One way to accomplish inhibition is bythe expression of RNA molecules such as those described herein, whichrepress the expression of PD-L1/L2 in the DCs transduced with a viralvector genome, such as the lentivirus vector genome, encoding one ormore of the relevant molecules. Maturation of DCs or expression ofparticular elements such as immune checkpoints, for example PD-1ligands, can be characterized by flow cytometry analysis ofup-regulation of surface marker such as MHC II, and by profilingexpressed chemokines and cytokines, for example, by performingtechniques and methods described herein.

A sequence encoding a detectable product, usually a protein, can beincluded to allow for identification of cells that are expressing thedesired immunogen. For example, a fluorescent marker protein, such asgreen fluorescent protein (GFP), is incorporated into the recombinantexpression construct along with a polynucleotide sequence of interest(i.e., encoding at least one immunogen). In other instances, the proteinmay be detectable by an antibody, or the protein may be an enzyme thatacts on a substrate to yield a detectable product, or may be a proteinproduct that allows selection of a transfected or transduced targetcell, for example confers drug resistance, such as hygromycinresistance. Typical selection genes encode proteins that conferresistance to antibiotics or other toxins suitable for use in eukaryoticcells, for example, neomycin, methotrexate, blasticidin, among othersknown in the art, or complement auxotrophic deficiencies, or supplycritical nutrients withheld from the media. The selectable marker canoptionally be present on a separate plasmid and introduced byco-transfection.

With respect to vector particles described herein, one or moremulticistronic expression units may be used that include two or more ofa polynucleotide sequence encoding an immunogen, and a sequence encodingan envelope molecule as described herein or one or more DC maturationfactors necessary for production of the desired vector particle inpackaging cells. The use of multicistronic vectors reduces the totalnumber of nucleic acid molecules required and thus may avoid thepossible difficulties associated with coordinating expression frommultiple vector genomes. In a multicistronic vector the various elementsto be expressed are operatively linked to one or more promoters (andother expression control elements as necessary). In some configurations,a multicistronic vector comprises a sequence encoding an at least oneimmunogen (i.e., one or more) of interest, a sequence encoding areporter product, and a sequence encoding one or more vector particlecomponents. In certain embodiments in which the recombinant constructcomprises a polynucleotide that encodes an immunogen, the constructoptionally encodes a DC maturation factor. In certain other embodiments,a multicistronic vector comprises a polynucleotide sequences that encodeeach of an immunogen, a DC maturation factor, and optionally viralcomponents when the expression vector is a viral expression vector. Instill other embodiments, multicistronic vectors direct expression andencode at least two or more immunogens.

Each component to be expressed in a multicistronic expression vector maybe separated, for example, by an internal ribosome entry site (IRES)element or a viral 2A element, to allow for separate expression of thevarious proteins from the same promoter. IRES elements and 2A elementsare known in the art (see, e.g., U.S. Pat. No. 4,937,190; de Felipe etal. 2004. Traffic 5: 616-626). In one embodiment, oligonucleotides suchas furin cleavage site sequences (RAKR) (see, e.g., Fang et al. 2005Nat. Biotech. 23: 584-590) linked with 2A-like sequences fromfoot-and-mouth diseases virus (FMDV); equine rhinitis A virus (ERAV);and thosea asigna virus (TaV) (see, e.g., Szymczak et al. 2004 Nat.Biotechnol. 22: 589-594) are used to separate genetic elements in amulticistronic vector. The efficacy of a particular multicistronicvector can readily be tested by detecting expression of each of thegenes using standard protocols.

In a specific exemplification, a viral vector genome comprises: acytomegalovirus (CMV) enhancer/promoter sequence; the R and U5 sequencesfrom the HIV 5′ LTR; a packaging sequence (Φ); the HIV-1 flap signal; aninternal enhancer; an internal promoter; a gene of interest; thewoodchuck hepatitis virus responsive element; a tRNA amber suppressorsequence; a U3 element with a deletion of its enhancer sequence; thechicken β-globin insulator; and the R and U5 sequences of the 3′ HIVLTR. In some exemplifications, the vector genome comprises an intactlentiviral 5′ LTR and a self-inactivating 3′ LTR (see, e.g., Iwakuma etal. Virology 15:120, 1999).

Construction of the vector genome can be accomplished using any suitablegenetic engineering techniques known in the art, including, withoutlimitation, the standard techniques of restriction endonucleasedigestion, ligation, transformation, plasmid purification, and DNAsequencing, for example as described in Sambrook et al. (1989 and 2001editions; Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY); Coffin et al. (Retroviruses. Cold Spring HarborLaboratory Press, N.Y. (1997)); and “RNA Viruses: A Practical Approach”(Alan J. Cann, Ed., Oxford University Press, (2000), each of theforegoing which is incorporated herein by reference in its entirety.

Vectors constructed for transient expression in mammalian cells may alsobe used. Transient expression involves the use of an expression vectorthat is able to replicate efficiently in a host cell, such that the hostcell accumulates many copies of the expression vector and, in turn,synthesizes high levels of a the polypeptide encoded by theimmunogen-specific polynucleotide in the expression vector. See Sambrooket al., supra, pp. 16.17-16.22, 1989. Other vectors and methods suitablefor adaptation to the expression of polypeptides are well known in theart and are readily adapted to the specific circumstances.

By using the teachings provided herein and the knowledge in the art, aperson skilled in the art will recognize that the efficacy of aparticular expression system can be tested by transfecting packagingcells with a vector comprising a polynucleotide sequence encoding areporter protein and measuring the expression using a suitabletechnique, for example, measuring fluorescence from a green fluorescentprotein conjugate. Other suitable reporter genes are well known in theart.

Exemplary Embodiments

In addition to any of the foregoing embodiments described in thedetailed description, embodiments are contemplated including any of thefollowing or any combinations thereof:

1. An immunogenic, pharmaceutical composition comprising,

(i) an envelope glycoprotein of HSV-2, or an immunological fragmentthereof;

(ii) a structural protein of HSV-2 other than an envelope glycoproteinof HSV-2, or an immunological fragment thereof;

(iii) optionally, an agent that activates innate immunity; and

(iv) a pharmaceutically acceptable carrier.

2. The composition of embodiment 1 wherein the envelope glycoprotein ofHSV-2 is gD2.

3. The composition of embodiment 1 comprising an immunological fragmentof the envelope glycoprotein gD2.

4. The composition of any of embodiments 1-3, wherein the structuralprotein of HSV-2 is selected from the group consisting of UL47, ICP0,UL19, UL25, UL46, UL39, UL7 and UL26.

5. The composition of embodiment 1 wherein the structural protein ofHSV-2 is UL19.

6. The composition of embodiment 2 wherein the structural protein ofHSV-2 is UL19.

7. The composition of embodiment 1 which comprises an immunologicalfragment of UL19.

8. The composition of embodiment 2 which comprises an immunologicalfragment of UL19, for example, SEQ ID NO 12.

9. The composition of embodiment 1 wherein the structural protein ofHSV-2 is UL25.

10. The composition of embodiment 2 wherein the structural protein ofHSV-2 is UL25.

11. The composition of embodiment 1 which comprises an immunologicalfragment of UL25.

12. The composition of embodiment 2 which comprises an immunologicalfragment of UL25.

13. The composition of embodiment 1 wherein the structural protein ofHSV-2 is UL47.

14. The composition of embodiment 2 wherein the structural protein ofHSV-2 is UL47.

15. The composition of embodiment 1 which comprises an immunologicalfragment of UL47.

16. The composition of embodiment 2 which comprises an immunologicalfragment of UL47.

17. The composition of any one of embodiments 1-16 further comprising asecond structural protein of HSV-2, or an immunological fragmentthereof.

18. The composition of embodiment 17 wherein the second structuralprotein of HSV-2 is selected from the group consisting of UL47, ICP0,UL19, UL25, UL46, UL39, UL7 and UL26, where the second structuralprotein is non-identical to the first structural protein.

19. The composition of embodiment 18 wherein the second structuralprotein is a full length protein.

20. The composition of embodiment 18 wherein the second structuralprotein is an immunological fragment of the second structural protein.

21. The composition of any of embodiments 5-8 further comprising UL25.

22. The composition of any of embodiments 5-8 further comprising animmunological fragment of UL25.

23. The composition of any of embodiments 5-8 further comprising UL47.

24. The composition of any of embodiments 5-8 further comprising animmunological fragment of UL47.

25. The composition of any of embodiments 9-12 further comprising UL19.

26. The composition of any of embodiments 9-12 further comprising animmunological fragment of UL19, for example, SEQ ID NO. 12.

27. The composition of any of embodiments 9-12 further comprising UL47.

28. The composition of any of embodiments 9-12 further comprising animmunological fragment of UL47.

29. The composition of any of embodiments 13-16 further comprising UL19.

30. The composition of any of embodiments 13-16 further comprising animmunological fragment of UL19, for example, SEQ ID NO. 12.

31. The composition of any of embodiments 13-16 further comprising UL25.

32. The composition of any of embodiments 13-16 further comprising animmunological fragment of UL25.

33. The composition of any of embodiments 1-32, wherein the agent is anadjuvant.

34. The composition of embodiment 33, wherein the adjuvant is GLA.

35. The composition of embodiment 1 comprising gD2; UL25; UL19; GLAadjuvant; and a pharmaceutically acceptable carrier.

36. The composition of embodiment 1 comprising gD2, UL25 and animmunological fragment of UL19, for example, SEQ ID NO. 12.

37. The composition of embodiment 1 comprising gD2, UL19, and animmunological fragment of UL25.

38. The composition of any of embodiments 35-37 further comprising UL47.

39. The composition of any of embodiments 35-37 further comprising animmunological fragment of UL47.

40. The composition of embodiment 1 comprising ICP0 or an immunologicalfragment thereof, and one or more of UL47 or an immunological fragmentthereof, UL19 or an immunological fragment thereof, UL25 or animmunological fragment thereof, UL46 or an immunological fragmentthereof, UL39 or an immunological fragment thereof, UL7 or animmunological fragment thereof, and UL26 or an immunological fragmentthereof.

41. The composition of embodiment 2 comprising ICP0 or an immunologicalfragment thereof, and one or more of UL47 or an immunological fragmentthereof, UL19 or an immunological fragment thereof, UL25 or animmunological fragment thereof, UL46 or an immunological fragmentthereof, UL39 or an immunological fragment thereof, UL7 or animmunological fragment thereof, and UL26 or an immunological fragmentthereof.

42. The composition of embodiments 40 or 41, comprising ICP0 or animmunological fragment thereof, and two additional structural proteinsor an immunological fragment thereof.

43. The composition of embodiment 1 comprising UL46 or an immunologicalfragment thereof and one or more of UL47 or an immunological fragmentthereof, UL19 or an immunological fragment thereof, UL25 or animmunological fragment thereof, ICP0 or an immunological fragmentthereof, UL39 or an immunological fragment thereof, UL7 or animmunological fragment thereof, and UL26 or an immunological fragmentthereof.

44. The composition of embodiment 2 comprising UL46 or an immunologicalfragment thereof and one or more of UL47 or an immunological fragmentthereof, UL19 or an immunological fragment thereof, UL25 or animmunological fragment thereof, ICP0 or an immunological fragmentthereof, UL39 or an immunological fragment thereof, UL7 or animmunological fragment thereof, and UL26 or an immunological fragmentthereof.

45. The composition of embodiments 43 or 44, comprising UL46 or animmunological fragment thereof, and two additional structural proteinsor an immunological fragment thereof.

46. A method for treating an HSV-2 infection in a subject, comprisingadministering the composition of any one of embodiments 1-45 to thesubject.

47. A method for generating an immune response to HSV-2 in a subject,comprising administering the composition of any one of embodiments 1-45to the subject.

48. The method of embodiment 47, wherein the subject is seropositive forHSV-2 and seropositive for HSV-1.

49. The method of embodiment 47, wherein the subject is seropositive forHSV-2 and seronegative for HSV-1.

50. A pharmaceutical composition comprising,

an antigenic portion of an envelope glycoprotein of HSV-2 and apharmaceutically acceptable carrier, where the antigenic portioncomprises a leader sequence of an envelope glycoprotein of HSV-2.

51. The composition of embodiment 50, wherein the antigenic portionbinds to neutralizing antibodies.

52. The composition of embodiment 50 wherein the envelope glycoproteinof HSV-2 is gD2 or gB2.

53. The composition of embodiments 50-52 wherein the antigenic portioncomprises two or more linear epitopes from the envelope glycoprotein.

54. The composition of embodiments 50-52 wherein the antigenic portioncomprises two or more discontinuous epitopes from the envelopeglycoprotein.

55. The composition of any of embodiments 50-54 further comprising anagent that activates innate immunity.

56. The composition of embodiment 55, wherein the agent is an adjuvant.

57. The composition of embodiment 56, wherein the adjuvant is GLA.

58. A method for treating an HSV-2 or HSV-1 infection in a subject,comprising administering the composition of any one of embodiments 50-57to the subject.

59. A method for generating an immune response to HSV-2 or HSV-1 in asubject, comprising administering the composition of any one ofembodiments 50-57 to the subject.

60. The method of embodiments 58-59, wherein the subject is seropositivefor HSV-2 and seropositive for HSV-1.

61. The method of embodiments 58-59, wherein the subject is seropositivefor HSV-2 and seronegative for HSV-1.

62. A kit comprising a vial comprising the composition of embodiment 50.

63. An isolated fragment of UL19 lacking at least amino acids 1-336 and1295-1374 of SEQ ID NO: 4.

64. An isolated polypeptide comprising a fragment of UL19 consisting ofSEQ ID NO: 12 or a fragment thereof.

65. The polypeptide of embodiment 64 further comprising a non-UL19peptide fused to the fragment of UL19.

66. An isolated polypeptide comprising a peptide that consists of anamino acid sequence at least 80% identical over 50 contiguous aminoacids of SEQ ID NO: 12, optionally fused to a non-UL19 peptide.

67. An immunogenic, pharmaceutical composition comprising,

(i) a polypeptide comprising the amino acid sequence of SEQ ID NO: 12 oran immunological variant or fragment thereof, or the fragment orpolypeptide of any of embodiments 63-67;

(ii) an adjuvant; and

(iii) a pharmaceutically acceptable carrier.

68. The composition of embodiment 67, wherein the adjuvant is a TLR4agonist.

69. The composition of embodiment 68, wherein the adjuvant is GLA (FIG.1).

70. The composition of embodiment 67 further comprising any one or moreof (a) an envelope protein of HSV-2, (b) a structural protein of HSV-2other than an envelope glycoprotein of HSV-2, or (c) an immunologicalfragment of (a) or (b).

71. The composition of embodiment 67, further comprising a structuralprotein of HSV-2.

72. The composition of embodiment 71, wherein the structural protein isselected from the group consisting of UL47, ICP0, UL25, UL46, UL39, UL7and UL26.

73. The composition of embodiment 67 further comprising gD2, or animmunological fragment thereof, UL25, or an immunological fragmentthereof, and optionally UL47, or an immunological fragment thereof.

74. An immunogenic, pharmaceutical composition comprising,

(i) an envelope glycoprotein of HSV-2, or an immunological fragmentthereof;

(ii) GLA (FIG. 1); and

(iii) a pharmaceutically acceptable carrier.

75. The composition of embodiment 74, wherein the envelope glycoproteinof HSV-2 or immunological fragment thereof is gD2 or immunologicalfragment thereof.

76. An immunogenic, pharmaceutical composition comprising,

(i) a structural protein of HSV-2 other than an envelope glycoprotein ofHSV-2, or an immunological fragment thereof;

(ii) GLA; and

(iii) a pharmaceutically acceptable carrier.

77. The composition of embodiment 76, wherein the structural protein ofHSV-2 or immunological fragment thereof is selected from the groupconsisting of UL47, ICP0, UL19, UL25, UL46, UL39, UL7 and UL26 or animmunological fragment of any of these.

78. The composition of any one of embodiments 33, 34, 56, 57, 66, 67,and 71-77, further comprising a second adjuvant.

79. The composition of embodiment 78, wherein the second adjuvant isselected from the group consisting of a TLR agonist, e.g. a TLR7 agonistor a TLR9 agonist; alum; an emulsion; a saponin; a cytokine; anunmethylated CpG dinucleotide; and a modified saponin.

80. The composition of embodiment 78, wherein the second adjuvant isselected from the group consisting of Freund's incomplete adjuvant,MF59™, Montanide™, AS02™, AS04™, QS-21™, and ISCOM™.

81. An immunogenic, pharmaceutical composition comprising,

(i) ICP4, or an immunological fragment thereof;

(ii) gD2, or an immunological fragment thereof;

(iii) GLA (FIG. 1); and

(iii) a pharmaceutically acceptable carrier.

82. An immunogenic, pharmaceutical composition comprising,

(i) an α group gene product of HSV-2, or an immunological fragmentthereof; and/or

(ii) a β1 gene product of HSV-2, or an immunological fragment thereof;

and/or

(iii) a β2 gene product of HSV-2, or an immunological fragment thereof;and/or

(iv) a γ1 gene product of HSV-2, or an immunological fragment thereof;and/or

(v) a 72 gene product of HSV-2, or an immunological fragment thereof;and/or

(vi) an adjuvant, preferably GLA (FIG. 1); and

(vii) a pharmaceutically acceptable carrier.

83. The composition of any one of embodiments 1-45, 50-57, and 65-82,further comprising a surfactant.

84. A method for treating an HSV-2 infection or an HSV-1 infection in asubject, comprising administering the composition of any one ofembodiments 65-83 to the subject.

85. A method for generating an immune response to HSV-2 or an HSV-1infection in a subject, comprising administering the composition of anyone of embodiments 65-83 to the subject.

86. The method of any one of embodiments 84-85, wherein the subject isseropositive for HSV-2 and seropositive for HSV-1.

87. The method of any one of embodiments 85-85, wherein the subject isseropositive for HSV-2 and seronegative for HSV-1.

88. The method of any one of embodiments 83-87 wherein theadministration route is intradermal, mucosal, intramuscular,subcutaneous, sublingual, rectal, or vaginal.

89. A method for reducing transmission of HSV-2 from a subject,comprising administering the composition of any one of embodiments 1-45,50-57, and 65-83 to the subject.

90. A method for reducing shedding of HSV-2 in a subject, comprisingadministering the composition of any one of embodiments 1-45, 50-57, and65-83 to the subject.

91. A method for reducing the frequency of lesions in a subject with anHSV-2 infection, comprising administering the composition of any one ofembodiments 1-45, 50-57, and 65-83 to the subject.

92. A method for reducing the risk of contracting HIV in a subject withan HSV-2 infection, comprising administering the composition of any oneof embodiments 1-45, 50-57, and 65-83 to the subject.

93. A method for inducing sterilizing immunity to HSV-2 in a subject,comprising administering the composition of any one of embodiments 1-45,50-57, and 65-83 to the subject.

94. A kit comprising the composition of any one of embodiments 1-45,50-57, and 65-83.

95. The kit of embodiment 94, further comprising an attenuated HSV1 orHSV2 virus.

96. The kit of embodiment 94, further comprising an inactivated HSV1 orHSV2 virus.

97. The kit of embodiment 94, further comprising a viral vectorcomprising a polynucleotide encoding an HSV1 or HSV2 antigen.

98. The kit of embodiment 94, further comprising a virus-like particlecomprising a polynucleotide encoding an HSV1 or HSV2 antigen.

99. The kit of embodiment 94, further comprising a polynucleotideencoding an HSV1 or HSV2 antigen.

100. The composition of any one of embodiments 1-45, wherein theenvelope glycoprotein and/or structural protein is fused to aheterologous peptide.

101. The method of any one of embodiments 58-61 and 84-93, furthercomprising administering a polynucleotide encoding an HSV1 and/or HSV2antigen.

102. The method of embodiment 101, wherein the polynucleotide is a partof the genome of a viral vector.

103. The method of any one of embodiments 58-61 and 84-93, furthercomprising administering an inactivated or attenuated HSV1 or HSV2virus.

104. The method of any one of embodiments 58-61 and 84-93, furthercomprising administering a virus-like particle comprising apolynucleotide encoding an HSV1 or HSV2 antigen.

105. An immunogenic, pharmaceutical composition comprising,

(i) a first polynucleotide encoding an envelope glycoprotein of HSV-2,or an immunological fragment thereof;

(ii) a second polynucleotide encoding a structural protein of HSV-2other than an envelope glycoprotein of HSV-2, or an immunologicalfragment thereof;

(iii) optionally an agent that activates innate immunity, such as anadjuvant; and

(iv) a pharmaceutically acceptable carrier.

106. The composition of embodiment 105 wherein the envelope glycoproteinof HSV-2 is gD2.

107. The composition of embodiment 105 comprising an immunologicalfragment of the envelope glycoprotein gD2.

108. The composition of any of embodiments 105-107, wherein thestructural protein of HSV-2 is selected from the group consisting ofUL47, ICP0, UL19, UL25, UL46, UL39, UL7 and UL26.

109. The composition of any of embodiments 105-107 wherein thestructural protein of HSV-2 or immunological fragment thereof is UL19 oran immunological fragment thereof.

110. The composition of any of embodiments 105-107 wherein the secondpolynucleotide encodes UL19.

111. The composition of any of embodiments 105-107 wherein the secondpolynucleotide encodes an immunological fragment of UL19, optionally thefragment or polypeptide of any one of embodiments 63-66.

112. The composition of any of embodiments 105-107 wherein the secondpolynucleotide encodes SEQ ID NO 12.

113. The composition of any of embodiments 105-107 wherein thestructural protein of HSV-2 or immunological fragment thereof is UL25 oran immunological fragment thereof.

114. The composition of any of embodiments 105-107 wherein the secondpolynucleotide encodes UL25.

115. The composition of any of embodiments 105-107 wherein the secondpolynucleotide encodes an immunological fragment of UL25.

116. The composition of any of embodiments 105-107 wherein thestructural protein of HSV-2 or immunological fragment thereof is UL47 oran immunological fragment thereof.

117. The composition of any one of embodiments 105-116 furthercomprising a third polynucleotide encoding a second structural proteinof HSV-2, or an immunological fragment thereof.

118. The composition of embodiment 117 wherein the second structuralprotein of HSV-2 is selected from the group consisting of UL47, ICP0,UL19, UL25, UL46, UL39, UL7 and UL26, and wherein the second structuralprotein is non-identical to the first structural protein.

119. The composition of embodiment 118 wherein the second structuralprotein is a full length protein or an immunological fragment thereof.

120. The composition of any of embodiments 100-112 further comprising apolynucleotide encoding UL25 or an immunological fragment thereof.

121. The composition of any of embodiments 106-109 further comprising apolynucleotide encoding UL47 or an immunological fragment thereof.

122. The composition of any of embodiments 113-115 further comprising apolynucleotide encoding UL19.

123. The composition of any of embodiments 113-115 further comprising apolynucleotide encoding SEQ ID NO. 12.

124. The composition of any of embodiments 113-115 further comprising apolynucleotide encoding UL47 or an immunological fragment thereof.

125. The composition of embodiment 116 further comprising apolynucleotide encoding UL19.

126. The composition of any of embodiments 116 further comprising apolynucleotide encoding SEQ ID NO. 12.

127. The composition of any of embodiments 116 further comprising apolynucleotide encoding UL25 or an immunological fragment thereof.

128. The composition of any of embodiments 105-127, wherein the agent isan adjuvant, optionally a TLR4 agonist.

129. The composition of embodiment 128, wherein the adjuvant is GLA.

130. The composition of embodiment 105, wherein the first polynucleotideencodes gD2; the second polynucleotide encodes UL25; and wherein thecomposition further comprises a third polynucleotide encoding UL19; GLAadjuvant; and a pharmaceutically acceptable carrier.

131. The composition of embodiment 105 wherein the first polynucleotideencodes gD2, the second polynucleotide encodes UL25, and wherein thecomposition further comprises a polynucleotide encoding SEQ ID NO. 12.

132. The composition of embodiment 105 wherein the first polynucleotideencodes gD2, the second polynucleotide encodes UL19, wherein thecomposition further comprises a polynucleotide encoding an immunologicalfragment of UL25.

133. The composition of any of embodiments 130-132 further comprising apolynucleotide encoding UL47 or an immunological fragment thereof.

134. The composition of embodiment 105 or 106 comprising apolynucleotide encoding ICP0 or an immunological fragment thereof, andone or more of a polynucleotide encoding UL47 or an immunologicalfragment thereof, UL19 or an immunological fragment thereof, UL25 or animmunological fragment thereof, UL46 or an immunological fragmentthereof, UL39 or an immunological fragment thereof, UL7 or animmunological fragment thereof, and UL26 or an immunological fragmentthereof.

135. The composition of embodiment 134, comprising a polynucleotideencoding ICP0 or an immunological fragment thereof, and two additionalstructural proteins or an immunological fragment thereof.

136. The composition of embodiment 105 or 106 comprising apolynucleotide encoding UL46 or an immunological fragment thereof andone or more of a polynucleotide encoding UL47 or an immunologicalfragment thereof, UL19 or an immunological fragment thereof, UL25 or animmunological fragment thereof, ICP0 or an immunological fragmentthereof, UL39 or an immunological fragment thereof, UL7 or animmunological fragment thereof, and UL26 or an immunological fragmentthereof.

138. The composition of embodiments 136, comprising a polynucleotideencoding UL46 or an immunological fragment thereof, and polynucleotidesencoding two additional structural proteins or an immunological fragmentthereof.

139. An immunogenic, pharmaceutical composition comprising,

(i) a first polynucleotide encoding a polypeptide comprising the aminoacid sequence of SEQ ID NO: 12 or an immunological variant or fragmentthereof;

(ii) optionally an agent that activates innate immunity, such as anadjuvant; and

(iii) a pharmaceutically acceptable carrier.

140. The composition of embodiment 139, wherein the agent is anadjuvant.

141. The composition of embodiment 140, wherein the adjuvant is GLA.

142. The composition of embodiment 139 further comprising a secondpolynucleotide encoding a structural protein of HSV-2 other than anenvelope glycoprotein of HSV-2, or an immunological fragment thereof.

143. The composition of embodiment 139, further comprising a thirdpolynucleotide encoding a structural protein of HSV-2 in addition toUL19(ud).

144. The composition of embodiment 143, wherein the structural proteinis selected from the group consisting of UL47, ICP0, UL25, UL46, UL39,UL7 and UL26.

145. An immunogenic, pharmaceutical composition comprising,

(i) a first polynucleotide encoding an envelope glycoprotein of HSV-2,or an immunological fragment thereof;

(ii) GLA; and

(iii) a pharmaceutically acceptable carrier.

146. The composition of embodiment 145, wherein the envelopeglycoprotein of HSV-2 is gD2.

147. An immunogenic, pharmaceutical composition comprising,

(i) a first polynucleotide encoding a structural protein of HSV-2 otherthan an envelope glycoprotein of HSV-2, or an immunological fragmentthereof;

(ii) GLA; and

(iii) a pharmaceutically acceptable carrier.

148. The composition of embodiment 147, wherein the structural proteinof HSV-2 is selected from the group consisting of UL47, ICP0, UL19,UL25, UL46, UL39, UL7 and UL26.

149. The composition of any one of embodiments 105149, furthercomprising a second adjuvant.

150. The composition of embodiment 149, wherein the second adjuvant isselected from the group consisting of a TLR agonist, alum, an emulsion,a saponin, a cytokine, an unmethylated CpG dinucleotide, and a modifiedsaponin.

151. The composition of embodiment 149, wherein the second adjuvant isselected from the group consisting of Freund's incomplete adjuvant,MF-59™, Montanide™, AS02™, AS04™, QS-21™, and ISCOM™.

152. An immunogenic, pharmaceutical composition comprising,

(i) a first polynucleotide encoding ICP4, or an immunological fragmentthereof;

(ii) a second polynucleotide encoding gD2, or an immunological fragmentthereof;

(iii) GLA; and

(iii) a pharmaceutically acceptable carrier.

153. An immunogenic, pharmaceutical composition comprising,

(i) a first polynucleotide encoding an immediate early gene product ofHSV-2, or an immunological fragment thereof;

(ii) a second polynucleotide encoding an early gene product of HSV-2, oran immunological fragment thereof;

(iii) a third polynucleotide encoding a late gene product of HSV-2, oran immunological fragment thereof; and

(iv) a pharmaceutically acceptable carrier.

154. The composition of any one of embodiments 105-153, furthercomprising a surfactant.

155. The composition of any one of embodiments 105-154, wherein thepolynucleotides are present in one or more recombinant expressionvectors.

156. The composition of embodiment 155, wherein the recombinantexpression vector is a viral vector or a virus-like particle.

157. A method for treating an HSV-2 or HSV-1 infection in a subject,comprising administering the composition of any one of embodiments105-156 to the subject and co-administering a second compositioncomprising an adjuvant.

158. The method of embodiment 157, wherein the adjuvant is a TLR4agonist.

159. The method of embodiment 158, wherein the TLR4 agonist is GLA.

160. A method for generating an immune response to HSV-2 or HSV-1 in asubject, comprising administering the composition of any one ofembodiments 105-156 to the subject and co-administering a secondcomposition comprising an adjuvant.

161. The method of embodiment 160, wherein the adjuvant is a TLR4agonist.

162. The method of embodiment 161, wherein the TLR4 agonist is GLA.

163. The composition of any one of embodiments 1-45, 50-57, and 66-83,further comprising a virus-like particle, wherein the virus-likeparticle comprises the envelope glycoprotein of HSV-2 or immunologicalfragment thereof and the structural protein of HSV-2 other than anenvelope glycoprotein of HSV-2 or immunological fragment thereof of anyone of embodiments 1-45; the antigenic portion of an envelopeglycoprotein of HSV-2 of any one of embodiments 50-57; the fragment ofUL19 of any one of embodiments 63-65; the polypeptide of any one ofembodiments 66-73; the envelope glycoprotein of HSV-2 or immunologicalfragment thereof of any one of embodiments 74-75; the structural proteinof any one of embodiments 76-77; or the ICP4 or immunological fragmentthereof and the gD2 or immunological fragment thereof of embodiment 81.

164. A method for treating an HSV-2 infection or an HSV-1 infection in asubject, comprising a priming step comprising administering anattenuated live HSV virus to the subject and a boosting step comprisingadministering the composition of any one of embodiments 1-45, 50-57,66-83 and 105-156 to the subject.

165. A method for generating an immune response to HSV-2 or an HSV-1infection in a subject, comprising a priming step comprisingadministering an attenuated live HSV virus to the subject and a boostingstep comprising administering the composition of any one of embodiments1-45, 50-57, 66-83 and 105-156 to the subject.

166. A method for treating an HSV-2 infection or an HSV-1 infection in asubject, comprising a priming step comprising administering thecomposition of any one of embodiments 1-45, 50-57, 66-83 and 105-156 tothe subject and a boosting step comprising administering an attenuatedlive HSV virus to the subject.

167. A method for generating an immune response to HSV-2 or an HSV-1infection in a subject, comprising a priming step comprisingadministering the composition of any one of embodiments 1-45, 50-57,66-83 and 105-156 to the subject and a boosting step comprisingadministering an attenuated live HSV virus to the subject.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Enhancement of CD4 T Cell-Based ImmunogenicityAgainst HSV-2 GD2 Protein when Formulated with the Adjuvant GLA-SEFollowing Multiple Vaccinations in Mice

In this example, the ability of GLA-SE to augment CD4 T cell responsesfollowing immunization of mice with a recombinant protein vaccine wasassessed.

Groups of five Balb/c mice were immunized via a prime/boost immunizationregimen (d0 prime/d21 boost) with either 0.8, 4, or 20 μg of recombinantgD protein in combination with either 0.8, 4, or 20 μg of GLA-SE (SEpercentage is 2% in this and all following studies), SE alone, or PBS,delivered intramuscularly in 100 μl (50 μl each leg). Mice immunizedwith GLA-SE, SE alone, or PBS in the absence of recombinant proteinserved as negative controls. Antigen-specific splenic CD4 T cellresponses were measured on day 4 post-boost by Intracellular CytokineStaining (ICS) for IFN-γ, TNF-α, and IL-2 after ex-vivo re-stimulationof splenocyte cultures for 5 hours with gD₂₇₂₋₂₈₅ peptide, which hadpreviously been identified as a CD4 T cell epitope in gD2 that ispresented in mice with the H-2d haplotype. As depicted in FIG. 2, a CD4T cell response to immunization with each dose of gD2 recombinantprotein was observed only when either GLA-SE or SE was included as anadjuvant. At each dose of recombinant gD2 antigen and at each dose ofGLA-SE, the magnitude of the gD2-specific CD4 T cell response wasincreased over the response observed to the same amount of recombinantgD2 antigen formulated with SE alone. In addition, the quality of theresponding antigen-specific CD4 T cell population, as measured by thefrequency of IFN-γ+, TNF-α+, and IL-2+ CD4 T cells (triple positive)within the responding CD4 T cell population was increased at each doseof recombinant gD2 protein and at each dose of GLA over that observedwhen gD2 was formulated with SE alone. The data from this study indicatethat the formulation of the adjuvant GLA-SE with recombinant HSV-2protein antigen substantially increases the performance of the vaccineover that which is achieved by immunizing with recombinant protein aloneor recombinant protein formulated with SE alone as measured by both themagnitude and quality of the cellular immune response.

Example 2 GLA Augments CD8 T Cell Responses 1N Mice

In this example, the ability of GLA-SE to augment CD8 T cell responseswas assessed following immunization of mice with a recombinant proteinvaccine.

Ovalbumin was used as a model protein. Female C57Bl/6 mice were injecteds.c. with lentivirus-encoding ovalbumin (“LV-OVA” in FIGS. 3 and 4) andboosted by i.m. injection on day 21 with recombinant ovalbuminadjuvanted with various doses of GLA-SE (“OVA+GLA/SE” in FIGS. 3 and 4).Four days later, splenic T cell responses were measured by intracellularcytokine staining (ICS) to the following in vitro stimulants: OVA MHCClass I peptides 55-62 and 257-264 and MHC Class II peptide 323-339, orantibodies to CD3 and to CD28. CD8 T cells are identified as thosesecreting any of the cytokines, IFN-γ,IL-2, and TNF-α

As shown in FIG. 3, there was a higher percentage of CD8 T cells in micethat received a boost of antigen, with the highest percentages in micethat received GLA-SE with the antigen in the boost. FIG. 4 providesexperimental detail of the ratios of four subsets of CD8 T cells.Therefore, an i.m. vaccine ‘boost’ with recombinant OVA protein+GLA-SEboosted pre-existing CD8 T cells that had been generated via previous LVvaccination. The mid (4 μg) and low (0.8 μg) doses of GLA provided thehighest increase of CD8 T cells under these experimental settings.Therefore, these data show that GLA adjuvanted protein can be used toboost a pre-existing CD8 memory T cell response specific for theprotein. Activation of CD8 memory cells is considered to be a desirableproperty of a therapeutic vaccine against HSV-2 for treatment ofinfected individuals, underscoring the superior properties GLAadjuvanted protein may confer to an HSV-2 vaccine.

Example 3

CD4 T Cell-Based Immunogenicity Against Individual HSV-2 GD2, UL19, andUL25 Proteins Following Multiple Vaccinations in Mice

The goal of this set of studies was to identify a single mouse strain inwhich the CD4 T cell-based immunogenicity against each protein subunitin the vaccine could be evaluated. To this end, a series of experimentswere conducted in mice to identify individual CD4 T cell epitopes withineach HSV-2 antigen (i.e. gD2, UL19, and UL25) within the context ofdifferent MHC haplotypes (i.e. BALB/c (H-2^(d)), C57BL/6 (H-2^(b)), andCB6F1 (H-2^(d)+2^(b))). The experimental strategy consisted of theimmunization of naïve mice with 5 μg of each recombinant protein antigenas a monovalent immunogen formulated with 5 μg GLA-SE intramuscularly in100 μl (50 μl each leg) within the context of a prime/boost immunizationregimen (d0 prime/d21 boost). Antigen-specific CD4 T cell responses wereanalyzed on day 4 post-boost using 15-mer peptide libraries (11 aaoverlap between peptides) whose sequence was derived from thecorresponding amino acid sequence of the monovalent immunogen. In theprimary screens, splenic CD4 cells were analyzed for the production ofIFN-γ, TNF-α, and IL-2 in response to the ex vivo simulation ofsplenocytic cultures with pools of individual 15-mer peptides from thepeptide library that corresponded to the individual HSV-2 encodedantigen. Observed CD4 T cell responses in the peptide pools wereconsidered to be positive hits, and secondary (and in some casestertiary) screens were subsequently conducted with an identicalimmunization and analysis strategies using either individual peptideswithin the positive pools from the previous screen as ex vivo stimulatesor peptides within the positive pools from the previous screen re-pooledin different combinations. As shown in FIGS. 5A-B, these studiesidentified individual 15-mer peptides against which an antigen-specificCD4 T cell response could be observed for each of the individualrecombinant HSV-2 proteins within the vaccine (i.e. gD2, UL19, and UL25)within the context of the MHC haplotype H-2b (C57BL/6 mice).

Example 4

CD4 T and B Cell-Based Immunogenicity Against Each Individual HSV-2Subunit Protein Following Multiple Vaccinations of a TrivalentFormulation in Mice

This example demonstrates the CD4 T cell and B cell-based immunogenicityagainst each of the individual recombinant protein subunits within thevaccine when they are delivered together as a trivalent formulation withGLA-SE in C57BL/6 mice. The experimental strategy consisted of using twogroups of five C57BL/6 mice. One group was immunized via a prime/boostimmunization regimen (d0 prime/d21 boost) with recombinant HSV-2 gD2,UL19, and UL25 proteins delivered in combination and formulated on anequi-molar basis (0.8, 3.3, and 1.4 μg of protein, respectively) incombination with 5.5 μg of GLA-SE delivered intramuscularly in 100 μl(50 μl each leg). The second group was mock immunized with vehicle(PBS). The animals were sacrificed on day 4 post-boost for theharvesting of the spleens and peripheral blood (via cardiac puncture).Antigen-specific splenic CD4 T cell responses were measured by ICS forIFNγ, TNFα, and IL-2 after the ex vivo re-stimulation of splenocytecultures with the 15-mer peptides previously identified as containingCD4 T cell epitopes for each recombinant protein immunogen within thetrivalent vaccine (see Example 3). The serum of each vaccinated and mockvaccinated mouse was analyzed for the presence of antigen-specificantibodies of the IgG1 subclass against each of the recombinant proteinimmunogens within the trivalent vaccine by direct ELISA. As shown inFIGS. 6A-B, antigen-specific CD4 T cell and antibody responses wereobserved to each of the HSV-2 recombinant protein antigens whendelivered together as a trivalent formulation with GLA-SE. These datasupport the significant immunogenicity of the trivalent vaccine and itsability to elicit a comprehensive immune response (both humoral andcellular) against HSV-2 proteins. Unexpectedly, the magnitude of theimmune responses generated were greatest for the UL19 antigen. UL19 hasnever been included as a component of any of the prior recombinantsubunit-based vaccines administered for the treatment or prevention ofHSV-2 infection in humans. These data provide evidence that the claimedvaccines display superior properties over the prior art vaccines.

Example 5 Antigen-Specific Cd4 T Cell Responses Following Single andMultiple Immunizations of HSV-2 UL19 with GLA-SE in Mice

This Example shows the CD4 T cell-based immunogenicity generated bysingle and repeat immunizations of HSV-2 UL19 formulated with GLA-SE inmice. For this study, two groups of five C57BL/6 mice received oneimmunization and two groups of five c57BL/6 mice received twoimmunizations (separated by 21 days) with 5 μg of recombinant UL19protein antigen as a monovalent immunogen with 5 μg GLA-SE. The groupsof mice were sacrificed at either day 4 or 10 after the finalimmunization for the analysis of antigen-specific CD4 T cell responses.The immunizations that the respective analysis groups received werestaggered in time such that all four groups of mice were sacrificed onthe same day for the analysis of the antigen-specific CD4 T cellresponse. The antigen-specific CD4 T cell response to the immunogen wasmeasured by the production of IFN-γ, TNF-α, and IL-2 in response to theex vivo stimulation of splenocytes with the individual UL19 15-merpeptides numbers 250 and 297 that had been previously identified ascontaining CD4 T cell epitopes specific for UL19 (see Example 3). Asdepicted in FIGS. 7A-B, at day four post-last immunization UL19-specificCD4 T cell responses were only detected in animals that received twoimmunizations, whereas UL19-specific CD4 T cell responses were detectedat day 10 post-last immunization within both the prime and theprime/boost arms of the experiment. At day 10 post-last immunization,the magnitude of the response was markedly increased (˜2.5 fold) in theanimals that received two immunizations as compared to those thatreceived only a single immunization. These findings show that repeatadministration of a vaccine containing a recombinant HSV-2protein+GLA-SE is a superior protocol for increasing the response andthe magnitude of the ensuing antigen-specific CD4 T cell response

To test the dependence of the increase in the CD4 T cell responsefollowing repeat administration of the vaccine on GLA-SE, a similarexperiment was performed in which groups of mice were immunized withUL19 protein alone or protein formulated with SE alone, or GLA-SE. Thegroups of mice were sacrificed at either day 5 or 10 post finalimmunization for the analysis of antigen-specific CD4 T cell responses.The antigen-specific CD4 T cell response to the immunogen was measuredby the production of IFN-γ, TNF-α, and IL-2 in response to the ex vivostimulation of splenocytes with the individual UL19 15-mer peptidesnumbers 250 and 297 that had been previously identified as containingCD4 T cell epitopes specific for UL19 (see Example 3). As depicted inFIGS. 8A-B, animals that received two immunizations as compared to thosethat received only a single immunization displayed a significantincrease in the antigen-specific CD4 T cell response, confirming theresults of the previous experiment. Importantly, this increase was foundto be dependent upon the GLA-SE adjuvant as mice receiving twoimmunizations displayed no significant CD4 T cell responses when theimmunogen was administered alone or with SE in the absence of GLA.

Example 6 Antigen-Specific Cd4 T Cell Responses Following Immunizationwith Trivalent HSV Vaccine Formulated with GLA-SE in Mice

This Example shows that CD4 T cell responses can be generated againsteach subunit of a trivalent subunit vaccine comprising the gD2, UL19,and UL25 antigens formulated in GLA-SE when the recombinant proteins areformulated on an equi-molar as well as an equi-mass basis. Groups offemale C57BL/6 mice (5 mice/group) were immunized with a trivalentvaccine wherein the total protein was either 5 μg or 15 μg on either anequi-molar or an equi-mass basis. Mice received a second immunizationwith a homologous formulation at day 21 and T cell responses weremeasured after ex vivo restimulation with an appropriate peptide by ICSfive days following the last immunization. As shown in FIG. 9,epitope-specific CD4 T cell responses are generated against eachindividual component of the trivalent HSV-2 subunit vaccine. Positiveresponses were observed despite whether the recombinant proteincomponents are formulated on an equi-molar or an equi-mass basis,indicating that the responses are not significantly impacted or alteredbased on relative protein composition of the vaccine.

Example 7 Enhancement of Antibody-Based Immunogenicity Against HSV-2 GD2Protein when Formulated with the Adjuvant GLA-SE Following MultipleVaccinations in Mice

In this example, the ability of GLA-SE to augment CD4 T cell responsesfollowing immunization of mice with a recombinant protein vaccine wasassessed.

Groups of five Balb/c mice were immunized via a prime/boost immunizationregimen (d0 prime/d21 boost) with 4 μg of recombinant gD protein incombination with either 4 μg of GLA-SE, SE alone, or PBS vehicle,delivered intramuscularly in 100 μl (50 μl per leg). HSV-2 gD2-specificantibodies of the IgG, IgG1, and IgG2a isotypes were measured by ELISA.As depicted in FIG. 10, GLA-SE adjuvant enhanced the total IgG responseagainst HSV-2 gD2, reduced the production of antigen-specific IgG1, andincreased the production of antigen-specific IgG2a.

Example 8 Enhancement of CD8 T Cell-Based Immunogenicity Against HSV-2UL19UD Protein when Formulated with the Adjuvant GLA-SE

In this example, the ability of GLA-SE to induce functional HSV-2UL19-specific CD8 T cell responses following immunization of mice with atrivalent vaccine containing recombinant HSV-2 gD2, UL19 upper domain(UL19ud; SEQ ID NO:12), and UL25 was assessed.

Groups of five C57BL/6 mice were given a single intramuscularimmunization of trivalent vaccine consisting of 514 each of recombinantgD2, UL19ud, and UL25 in combination with either 5 μg GLA-SE or 5%dextrose vehicle. Mice immunized with vehicle alone served as negativecontrols. Antigen-specific splenic CD4 and CD8 T cell responses weremeasured on day 6 post-immunization by Intracellular Cytokine Staining(ICS) for IFN-γ, TNF-α, and IL-2 after ex-vivo re-stimulation ofsplenocyte cultures for 5 hours with gD2, UL19, or UL25 peptides. Asdepicted in FIG. 11, panel A, a CD4 T cell response to each component ofthe trivalent vaccine (gD2, UL19ud, and UL25) was observed when GLA-SEwas included as an adjuvant. Notably, as depicted in FIG. 11, panel B, aCD8 T cell response was observed against the UL19ud antigen when givenwith GLA-SE. Confirming that these CD8 T cells are functional, mice thatwere unimmunized or immunized 4 weeks earlier with trivalent vaccinewith GLA-SE were challenged subcutaneously with attenuated HSV-2thymidine kinase-deficient (TK-) virus and UL19-specific CD8 T cellresponses were measured by ICS. As depicted in FIG. 11, panel C, themagnitude of the CD8 T cell response upon viral challenge was greater inmice previously immunized with vaccine.

Example 9 Enhancement of Prophylactic Antiviral Efficacy of RecombinantHSV-2 Protein Vaccine when Formulated with the Adjuvant GLA-SE

In this example, the ability of GLA-SE to enhance the ability of abivalent recombinant HSV-2 protein vaccine to protect against lethalHSV-2 challenge was assessed.

Groups of ten C57BL/6 mice were given two intramuscular immunizations,separated by 28 days, of bivalent vaccine consisting of 5 μg each ofrecombinant gD2 and UL19ud in combination with either 5 μg GLA-SE or 5%dextrose vehicle. Mice immunized with 5 μg GLA-SE alone served asnegative controls. 22 days after the second immunization, mice weretreated with depot medroxyprogesterone acetate and then challenged sixdays later with a 50×LD₅₀ dose of wild-type HSV-2 intravaginally. Micemonitored daily for formation of genital lesions and survival. On days1, 3, and 5 post infection, vaginal swabs were collected forquantitation of HSV-2 DNA by PCR. Approximately 2 months post infection,the dorsal root ganglia were harvested from surviving mice and latentHSV-2 DNA was quantified by PCR. As depicted in FIG. 12, panel A, miceimmunized with gD2 and UL19ud with GLA-SE has dramatically reducedlesion formation and increased survival compared to mice immunized witheither gD2 and UL19ud alone or GLA-SE alone. Likewise, as depicted inFIG. 12, panel B, 9 out of 10 mice immunized with gD2 and UL19ud withGLA-SE had no detectable HSV-2 DNA by day 5, whereas mice in eithercontrol group showed sustained levels of HSV-2 in the vagina through day5. As depicted in FIG. 12, panel C, though there were three survivors inthe GLA-SE only group, 2 out of 3 of these mice showed significantlevels of latent HSV-2 in the dorsal root ganglia, mice immunized withgD2 and UL19ud with GLA-SE showed little to no detectable HSV-2 in theganglia.

Example 10 Enhancement of Expansion of Pre-Existing Memory Cd8 T Cellsby Recombinant HSV-2 Protein Vaccine when Formulated with the AdjuvantGLA-SE

In this example, the ability of GLA-SE to enhance the ability of atrivalent recombinant HSV-2 protein vaccine to expand memory CD8 T cellspreviously induced by HSV-2 infection was assessed.

Groups of five C57BL/6 mice were infected subcutaneously with asublethal dose of attenuated HSV-2 thymidine kinase-deficient (TK-)virus. 28 days later, infected or uninfected mice were immunized with atrivalent vaccine consisting of 5 μg each of recombinant gD2, UL19ud(SEQ ID NO:12), and UL25 in combination with 5 μg GLA-SE or 5% dextrosevehicle. Control groups included infected mice treated with GLA-SE aloneor vehicle alone, as well as naïve mice treated with vehicle alone. Sixdays post immunization, UL19-specific CD4 and CD8 T cell responses weremeasured by ICS. As depicted in FIG. 13, the frequency of UL19-specificCD4 and CD8 T cells was greater after immunization of previouslyinfected mice, indicating that there was recall of infection-inducedmemory T cells. Importantly, maximum expansion of these memory T cellsby recombinant protein vaccine required the presence of GLA-SE adjuvant.

Example 11 Ability of a Recombinant HSV-2 Protein Vaccine to TreatRecurrent HSV-2 in Guinea Pigs

In this example, the ability of a trivalent recombinant HSV-2 proteinvaccine to reduce the frequency of recurrent HSV-2 lesions was assessed.

Groups of seven guinea pigs infected intravaginally with a sublethaldose of HSV-2 strain 333 virus. On days 13 and 27 post infection, guineapigs were immunized with a trivalent vaccine consisting of 5 μg each ofrecombinant gD2, UL19ud (see SEQ ID NO:12), and UL25 in combination with5 μg GLA-SE. Infected guinea pigs treated with GLA-SE alone served asnegative controls. Animals were monitored daily for vaginal lesions andscores of 0-4 were assigned for each lesion day. Daily lesions scores ineach group were averaged and plotted versus time. As depicted in FIG.14, animals treated with trivalent vaccine plus GLA-SE had approximatelya 50% reduction in recurrent lesions compared to animals treated withGLA-SE alone.

Example 12 Construction of Immunogenic Protein Derived from HSV-2Envelope Glycoprotein and Containing a Leader Sequence

In this example, an immunogenic protein is constructed from gD2 sequenceand comprises the gD2 leader sequence.

The leader sequence of gD2 is 40 amino acids long (residues 1-40 in SEQID No.: 1). A nucleotide sequence encoding a 100 amino acid fragment(residues 1-100) is inserted into an expression vector. Site-directedmutagenesis is used to change residues 38-42 from CysAlaLysTyr (SEQ IDNO: 16) to GlyLeuAlaVal (SEQ ID NO: 17) or other sequence that isn'tcleaved during protein synthesis. CHO cells are transformed with thevector containing the altered sequence and gD2 protein is isolated.Alternatively, the nucleotide sequence is inserted into a baculovirusexpression vector and protein isolated from Sf9 cells. Verification thatthe leader sequence is present is obtained by HPLC analysis.

Example 13 Protective Efficacy of GLA/SE Plus Recombinant TrivalentProtein Vaccine Against Lethal Challenge with Virulent HSV-2

In this example, the ability of a trivalent recombinant HSV-2 proteinvaccine plus GLA adjuvant to protect against lethal HSV-2 was assessed.

Groups of ten C57BL/6 mice were given two intramuscular immunizations,separated by 28 days, of trivalent vaccine consisting of 5 μg each ofrecombinant gD2, UL19ud (see SEQ ID NO:12) and UL25 in combination witheither 5 μg GLA-SE or 5% dextrose vehicle. Mice immunized with 5 μgGLA-SE alone served as negative controls. An additional control groupconsisted of mice immunized with 5 μg GLA-SE and 1 milligram per ml ofaciclovir (ACV) in the drinking water starting 24 hours after challenge.Twenty-two days after the second immunization, mice were treated withdepot medroxyprogesterone acetate and then challenged six days laterwith a 50×LD₅₀ dose of wild-type HSV-2 intravaginally. Mice monitoreddaily for formation of genital lesions and survival. On days 1, 3, and 5post infection, vaginal swabs were collected for quantitation of HSV-2DNA by PCR.

As depicted in FIG. 15, mice immunized with trivalent recombinant gD2,UL19ud and UL25 with GLA-SE have dramatically reduced lesion formation(panel A) and have increased survival (panel B) compared to miceimmunized with either trivalent protein vaccine alone or GLA-SE alone.Likewise, as depicted in FIG. 16, 7 out of 10 mice immunized withgD2/UL19ud/UL25 with GLA-SE had no detectable vaginal HSV-2 DNA by day5, whereas mice in all three control groups showed sustained levels ofHSV-2 in the vagina through day 5. The animals that received acycloviralso had the same high HSV-2 DNA viral loads on days 1, 3, and 5. Theanimals that received the active vaccine of GLA/SE plus gD2/UL19ud/UL25had notably lower viral loads, with many animals sterilizing (i.e., nodetectable viral loads) by day 5.

In summary, these experiments demonstrate in vivo protective efficacy ofGLA/SE+ recombinant trivalent gD2/UL19ud/UL25 protein vaccine againstlethal challenge with virulent HSV-2.

Example 14 Safety and Immunogenicity of Vaccine in Humans

The safety and immunogenicity of immunogens described above formulatedwith GLA-SE, or SE alone may be tested in a Phase 1A/1B study designusing HSV-2 seronegative subjects (target for prophylactic vaccine) andHSV-2 seropositive subjects (target for immunotherapeutic vaccine). Thestudy design may follow that established by the HIV Vaccine TrialsNetwork (HVTN), and has been used in 40 human HIV-1 phase IA vaccinetrials in the last 10 years.

The design of these Phase 1A trials consists of a standardized format of12 subjects per group (10 vaccine—2 placebo) and is based upon theability to define a serious adverse event at a 15% prevalence. Vaccinesthat are not immunogenic (<2 of 10 subjects develop immunity) are alsodefined. In the HSV-2 Phase 1A study, subjects receive 3 i.m.immunizations of 1 μg or 2.5 μg GLA-SE at 4 week intervals. A total of48 HSV seronegative and HSV-2 seropositive subjects (HSV-1 seropositiveor HSV-1 seronegative) are immunized in the Phase 1A trial.

HSV-2 seronegative subjects are defined by Western Blot at Day 0. Inaddition to safety assessments, subjects on study may be monitored for apossible vaccine-induced HSV-2 specific immune humoral and cellularimmune response, and frequency of recurrence of genital ulcers (HSV-2seropositive subjects only). For the HSV-2 infected population, twopre-vaccination time points may be used to establish antibody to gD2.Cellular immunity to HSV-2 recombinant proteins may be assessed by IFN-γELISPOT and ICS assays, and gD2-specific humoral immunity by ELISA andneutralizing antibody assays.

From the foregoing it will be appreciated that, although specificembodiments have been described herein for purposes of illustration,various modifications may be made without deviating from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

What is claimed is:
 1. A trivalent immunogenic composition comprising:(a) an immunogenic fragment of an Herpes Simplex virus type 2 (HSV-2)UL19 polypeptide wherein the immunogenic fragment of UL19 has the aminoacid sequence set forth in SEQ ID NO: 12, and wherein the immunogenicfragment lacks at least 75% of amino acids 1-450 of SEQ ID NO: 4 andlacks at least 75% of amino acids of 1055-1374 of SEQ ID NO: 4; or animmunogenic variant thereof that retains at least 90% amino acididentity over the full length of the immunogenic fragment, glycoproteinD of HSV-2 (gD2), wherein the gD2 comprises the amino acid sequence setforth in SEQ ID NO: 2 or 3, or an immunogenic fragment thereofcomprising at least 15 contiguous amino acids of SEQ ID NO: 2 or 3, andUL25, wherein the UL25 comprises the amino acid sequence set forth inSEQ ID NO: 5 or an immunogenic fragment thereof comprising at least 15contiguous amino acids of SEQ ID NO: 5; (b) an amount of GlucopyranosylLipid Adjuvant (GLA) that increases the immune response; and (c) apharmaceutically acceptable carrier.
 2. The trivalent composition ofclaim 1, wherein the GLA is in the form of an oil-in-water emulsion oris in an aqueous form.
 3. The trivalent composition of claim 2, whereinthe oil-in-water emulsion comprises squalene.
 4. A method of generatingan immune response in a subject comprising administering the compositionof any one of claims 1, 2 or 3 to the subject.
 5. The method of claim 4wherein the administration route is intradermal, mucosal, intramuscular,subcutaneous, sublingual, rectal, or vaginal.
 6. The method of any oneof claims 4 or 5 further comprising administering a second, third orfourth composition according to any one of claims 1, 2 or 3 to thesubject.
 7. The trivalent composition of claim 1, wherein theimmunogenic fragment of UL19 has the amino acid sequence set forth inSEQ ID NO:12.
 8. The trivalent composition of claim 1, wherein theimmunogenic fragment of UL19 is fused to a heterologous peptide.
 9. Thetrivalent composition of claim 8, wherein the heterologous peptide is atag or a marker sequence.
 10. The trivalent composition of claim 9,wherein the tag is a histidine tag which optionally comprises a cleavagesequence.
 11. The trivalent composition of claim 1, wherein the UL25comprises the amino acid sequence set forth in SEQ ID NO: 5, or avariant thereof that retains at least 90% amino acid identity over thefull length of SEQ ID NO: 5 and is capable of inducing an immuneresponse against the UL25 protein set forth in SEQ ID NO: 5, or animmunogenic fragment thereof comprising at least 15 contiguous aminoacids of SEQ ID NO:
 5. 12. The trivalent composition of claim 1, whereinthe gD2 comprises the amino acid sequence set forth in SEQ ID NO:2 or 3,or a variant thereof that retains at least 90% amino acid identity overthe full length of SEQ ID NO: 2 or 3 and is capable of inducing animmune response against the gD2 protein set forth in SEQ ID NO:2 or 3,or an immunogenic fragment thereof comprising at least 15 contiguousamino acids of SEQ ID NO: 2 or
 3. 13. The trivalent composition of claim1, wherein the UL25 comprises the amino acid sequence set forth in SEQID NO:5, or a variant thereof that retains at least 90% amino acididentity over the full length of SEQ ID NO: 5 and is capable of inducingan immune response against the UL25 protein set forth in SEQ ID NO:5, oran immunogenic fragment thereof comprising at least 15 contiguous aminoacids of SEQ ID NO: 5; and wherein the gD2 comprises the amino acidsequence set forth in SEQ ID NO:2 or 3, or a variant thereof thatretains at least 90% amino acid identity over the full length of SEQ IDNO: 2 or 3 and is capable of inducing an immune response against the gD2protein set forth in SEQ ID NO: 2 or 3, or an immunogenic fragmentthereof comprising at least 15 contiguous amino acids of SEQ ID NO: 2 or3.
 14. The trivalent composition of claim 1, wherein gD2 is fused to aheterologous peptide, or UL25 is fused to a heterologous peptide, orboth.